Eradication of bacterial biofilm using anti-amyloid monoclonal antibodies

ABSTRACT

The present invention features compositions comprising an anti-amyloid antibody and methods of treating microbial infection and treating or preventing microbial biofilms using the composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/676,390, filed May 25, 2018 which is hereby incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI132996 awardedby the National Institutes of Health (NIH). The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Bacterial biofilms are of significant relevance and are oftendeleterious in the medical setting. Shielded within a denseextracellular matrix, bacterial biofilms are highly resistant toantibiotics, antimicrobials and responses generated by innate immunecells (Thurlow et al., 2011, J Immunol, 186 (11):6585-96). The treatmentstrategies to eradicate biofilms are limited. The current treatmentstrategy to eliminate biofilm associated infections is through the useof antibiotics. In comparison to planktonic bacteria, 100 to 1000 foldgreater concentration of antibiotics is required to combat biofilmassociated infections (Anwar and Costerton, 1990, Antimicrob AgentsChemother, 34 (9):1666-71, Moskowitz et al., 2004, J Clin Microbiol, 42(5):1915-22). Within biofilms, bacteria are slow growing or persistentrendering most antibiotics that target cellular biology or bacterialreplication ineffective (Keren et al., 2004, FEMS Microbiol Lett, 230(1):13-8, Brown et al., 1988, J Antimicrob Chemother, 22 (6):777-80,Stewart, 2002, Int J Med Microbiol, 292 (2):107-13, Lewis, 2001,Antimicrob Agents Chemother, 45 (4):999-1007). As more than 65% ofinfections are due to bacterial biofilms (Larsen et al., 2007, EnvironMicrobiol, 9 (12):3077-90, Costerton et al., 1999, Science, 284(5418):1318-22), novel treatments to disrupt bacterial biofilms isnecessary. Disruption of the biofilm matrix would enhance thesusceptibility of the biofilm to both innate immune system as well asantibiotic treatment thus promoting resolution of the biofilm.

A major proteinaceous component of enteric biofilms is amyloid curli.Amyloid curli is the most well characterized bacterial amyloid expressedwithin biofilms (Hung et al., 2013, MBio, 4 (5):e00645-13). Curli isspecifically expressed by bacteria of the Enterobacteriaceae family,although approximately 40% of bacterial species produce amyloids as amajor component of biofilms (Larsen et al., 2007, Environ Microbiol, 9(12):3077-90). Defined by its hallmark beta sheet structure where theβ-sheets are perpendicular to the fiber axis (Sunde et al., 1997, J MolBiol, 273 (3):729-39; Nelson et al., 2005, Nature, 435 (7043):773-8;Sunde et al., 1997, Adv Protein Chem, 50:123-59), curli is expressed bythe bidirectional curli specific gene csgBAC and csgDEFG operons(Chapman et al., 2002, Science, 295 (5556):851-5). The production ofcurli is a highly regulated process. When grown under stressfulconditions, activation of the csg genes leads the production of the maincurli proteins (along with other accessory proteins), CsgA and CsgB(Chapman et al., 2002, Science, 295 (5556):851-5; Zhou et al., 2012,Methods Mol Biol, 849:303-20). With the aid of other accessory proteins,CsgA is produced as a monomeric unit and is secreted extracellular whereit fibrilizes into the mature curli amyloid fibril (Robinson et al.,2006, Mol Microbiol, 59 (3):870-81). CsgB aids in the nucleation of CsgAmonomers and attaches CsgA to the cell surface (White et al., 2001, JMol Biol, 311 (4):735-49; Hammer et al., 2007, Proc Natl Acad Sci USA.,104 (30):12494-9).

Curli has numerous functions within the enteric biofilm. Curli serves asa scaffold of which allows the formation of the mature three dimensionalbiofilm (Reisner et al., 2003, Mol Microbiol, 48 (4):933-46; Costertonet al., 1995, Annu Rev Microbiol, 49:711-45). Forming a mesh likenetwork, curli enshrouds the bacteria creating a protective capsule.Curli expressed by the single bacterium promotes adhesion of multiplebacteria within the biofilm as well as aiding in surface attachment(Kikuchi et al., 2005, Microbiol Immunol, 49 (9):875-84). The productionof other integral biofilm components of S. typhimurium, such ascellulose, are dependent of the production of curli. Amyloids are highlyresistant to proteolytic and chemical degradation. Amyloids such ascurli need to be exposed 90 percent formic acid or hexafluoroisopropanol(HFIP) to depolymerize the fibril into monomeric subunits (Zhou et al.,2013, Methods Mol Biol, 966:53-75). Without the production of curli,biofilms of S. typhimurium are destabilized and fail to form maturebiofilms (Kikuchi et al., 2005, Microbiol Immunol, 49 (9):875-84).

Bacterial biofilms are frequently associated with infections and aredifficult to eradicate. Further, there is no treatment for clearingbacterial biofilms that form on indwelling medical device (catheters,heart valves, artificial joints, etc.), so that the foreign objectsusually need to be removed and/or replaced, at great cost and morbidityto the patient. Biofilms are also important components of chronicbacterial wound infections.

Thus, there is a need in the art for methods for preventing biofilmformation as well as for disrupting formed biofilms for treatment andprevention of microbial infections. The current invention satisfies thisunmet need.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a composition comprising atherapeutic antibody wherein the antibody is specific for binding to anepitope of curli and further wherein the epitope of curli comprises asequence having homology to an antibody binding site of one or moreheterologous amyloid proteins.

In one embodiment, the antibody inhibits fibrillization of one or moreheterologous amyloid proteins. In embodiment, the antibody inhibitsfibrillization of amyloid-β.

In one embodiment, the antibody prevents biofilm formation or altersbiofilm architecture. In one embodiment, the antibody is effective inreducing biofilm mass. In one embodiment, the biofilm mass is associatedwith a gram-positive bacteria, a gram-negative bacteria, or acombination thereof.

In one embodiment, the antibody is ALZ.3H3, ALZ.2C10, ALZ.4G1, orALZ.4A6.

In one embodiment, the antibody inhibits amyloid-β fibrillization andprevents biofilm formation or alters biofilm architecture. In oneembodiment, the antibody is an ALZ.3H3 antibody. In one embodiment theALZ.3H3 antibody comprises at least one of a heavy chain amino acidsequence as set forth in SEQ ID NO:2 and a light chain amino acidsequence as set forth in SEQ ID NO:35. In one embodiment the ALZ.3H3antibody comprises at least one of a heavy chain amino acid sequenceencoded by a nucleotide sequence as set forth in SEQ ID NO:1 and a lightchain amino acid sequence encoded by a nucleotide sequence as set forthin SEQ ID NO:34.

In one embodiment, the antibody prevents biofilm formation or altersbiofilm architecture. In one embodiment, the antibody is an ALZ.4G1antibody. In one embodiment the ALZ.4G1 antibody comprises at least oneof a heavy chain amino acid sequence as set forth in SEQ ID NO:61 and alight chain amino acid sequence as set forth in SEQ ID NO:63. In oneembodiment the ALZ.4G1 antibody comprises at least one of a heavy chainamino acid sequence encoded by a nucleotide sequence as set forth in SEQID NO:60 and a light chain amino acid sequence encoded by a nucleotidesequence as set forth in SEQ ID NO:62.

In one embodiment, the antibody inhibits amyloid-β fibrillization. Inone embodiment, the antibody is an ALZ.4A6 antibody. In one embodimentthe ALZ.4A6 antibody comprises at least one of a heavy chain amino acidsequence as set forth in SEQ ID NO:57 and a light chain amino acidsequence as set forth in SEQ ID NO:59. In one embodiment the ALZ.4A6antibody comprises at least one of a heavy chain amino acid sequenceencoded by a nucleotide sequence as set forth in SEQ ID NO:56 and alight chain amino acid sequence encoded by a nucleotide sequence as setforth in SEQ ID NO:58

In one embodiment, the composition is for application to a surface of amedical device.

In one embodiment, the composition comprises an antibiotic.

In one embodiment, the composition comprises one or morepharmaceutically acceptable carriers or excipients.

In one embodiment, the formulation is a topical formulation in the formof a cream, a lotion, an ointment, a hydrogel, a colloid, a gel, a foam,an oil, a milk, a suspension, a wipe, a sponge, a solution, an emulsion,a paste, a patch, a pledget, a swab, a dressing, a spray or a pad.

In one embodiment, the invention relates to a method of decolonizing amicrobial organism comprising contacting the microbial organism with acomposition comprising a therapeutic antibody, wherein the antibody isspecific for binding to an epitope of curli and further wherein theepitope of curli comprises a sequence having homology to an antibodybinding site of one or more heterologous amyloid proteins. In oneembodiment, the antibody the antibody inhibits fibrillization of one ormore heterologous amyloid proteins, prevents biofilm formation, altersbiofilm architecture, or any combination thereof. In one embodiment, theantibody is ALZ.3H3, ALZ.4G1, ALZ.2C10 or ALZ.4A6.

In one embodiment, the antibody inhibits amyloid-β fibrillization andprevents biofilm formation or alters biofilm architecture. In oneembodiment, the antibody is an ALZ.3H3 antibody. In one embodiment theALZ.3H3 antibody comprises at least one of a heavy chain amino acidsequence as set forth in SEQ ID NO:2 and a light chain amino acidsequence as set forth in SEQ ID NO:35. In one embodiment the ALZ.3H3antibody comprises at least one of a heavy chain amino acid sequenceencoded by a nucleotide sequence as set forth in SEQ ID NO:1 and a lightchain amino acid sequence encoded by a nucleotide sequence as set forthin SEQ ID NO:34.

In one embodiment, the antibody prevents biofilm formation or altersbiofilm architecture. In one embodiment, the antibody is an ALZ.4G1antibody. In one embodiment the ALZ.4G1 antibody comprises at least oneof a heavy chain amino acid sequence as set forth in SEQ ID NO:61 and alight chain amino acid sequence as set forth in SEQ ID NO:63. In oneembodiment the ALZ.4G1 antibody comprises at least one of a heavy chainamino acid sequence encoded by a nucleotide sequence as set forth in SEQID NO:60 and a light chain amino acid sequence encoded by a nucleotidesequence as set forth in SEQ ID NO:62.

In one embodiment, the antibody inhibits amyloid-β fibrillization. Inone embodiment, the antibody is an ALZ.4A6 antibody. In one embodimentthe ALZ.4A6 antibody comprises at least one of a heavy chain amino acidsequence as set forth in SEQ ID NO:57 and a light chain amino acidsequence as set forth in SEQ ID NO:59. In one embodiment the ALZ.4A6antibody comprises at least one of a heavy chain amino acid sequenceencoded by a nucleotide sequence as set forth in SEQ ID NO:56 and alight chain amino acid sequence encoded by a nucleotide sequence as setforth in SEQ ID NO:58

In one embodiment, the invention relates to a method of destroying ordisrupting or inhibiting or reducing biofilm formation of a microbialorganism comprising contacting the microbial organism with a compositioncomprising a therapeutic antibody, wherein the antibody is specific forbinding to an epitope of curli and further wherein the epitope of curlicomprises a sequence having homology to an antibody binding site of oneor more heterologous amyloid proteins. In one embodiment, the antibodythe antibody inhibits fibrillization of one or more heterologous amyloidproteins, prevents biofilm formation, alters biofilm architecture, orany combination thereof. In one embodiment, the microbial organism is abacterium.

In one embodiment, the invention relates to a method of treating amicrobial infection in a subject comprising administering to the subjecta therapeutically effective amount of a composition comprising atherapeutic antibody, wherein the antibody is specific for binding to anepitope of curli and further wherein the epitope of curli comprises asequence having homology to an antibody binding site of one or moreheterologous amyloid proteins. In one embodiment, the antibody theantibody inhibits fibrillization of one or more heterologous amyloidproteins, prevents biofilm formation, alters biofilm architecture, orany combination thereof. In one embodiment, the antibody is ALZ.3H3,ALZ.4G1, ALZ.2C10 or ALZ.4A6.

In one embodiment, the microbial infection is a bacterial infection. Inone embodiment, the bacterial infection is characterized by colonizationof a bacterium. In one embodiment, the bacterial infection ischaracterized by biofilm formation.

In one embodiment, the microbial infection is a topical infection. Inone embodiment, the topical infection is a wound, ulcer or lesion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A through FIG. 1C depict exemplary experimental resultsdemonstrating that the monoclonal antibody ALZ.3H3 disrupts S.typhimurium biofilm architecture and integrity. FIG. 1A depictsexemplary experimental results demonstrating that the architecture of S.typhimurium biofilm is altered with different treatments. FIG. 1Bdepicts exemplary images used to quantify the particles in the biofilm.FIG. 1C depicts an exemplary graph demonstrating the numbers ofparticles in the disperse portion of the biofilms (greater than 20 μmfrom the surface) in FIG. 1A.

FIG. 2A through FIG. 2C depict exemplary experimental resultsdemonstrating that the monoclonal antibody ALZ.3H3 alters biofilmintegrity of pre-established S. typhimurium biofilms. FIG. 2A depicts aschematic demonstrating the experimental design. FIG. 2B depictsexemplary experimental results demonstrating the S. typhimurium biofilmarchitecture with each treatment. FIG. 2C depicts an exemplary graphdemonstrating the numbers of particles in the disperse portion of thebiofilms (greater than 20 μm from the surface) in FIG. 2B.

FIG. 3 depicts a crystal violet assay dose curve of ALZ.3H3.

FIG. 4A and FIG. 4B depict analyses of biofilms of S. typhimurium. FIG.4A depicts a confocal analysis of biofilms of S. typhimurium grown inthe presence (10 ug/ml, 25 ug/ml, 50 ug/ml, 250 ug/ml, 500 ug/ml) orabsence (untreated) of 3H3. Biofilms were stained with syto9 (greennucleic acid stain for bacteria) and amyloid dye Congo red (red curli).Biofilms imaged using Leica TCS confocal at 63×. Biofilm 3Dreconstructions created using ImageJ 3D viewer software. Scale barrepresents 50 um. FIG. 4B depicts the biofilm thickness (um) of biofilmsof S. typhimurium grown in the presence (10 ug/ml, 25 ug/ml, 50 ug/ml,250 ug/ml, 500 ug/ml) or absence (untreated) of 3H3. Thickness measuredusing Leica TCS confocal microscopy software.

FIG. 5A through FIG. 5B depict exemplary experimental resultsdemonstrating that ALZ.3H3 reduces fibrillization of curli. FIG. 5Adepicts exemplary experimental results demonstrating that pan-amyloidantibodies prevent fibrillization in a Thioflavin T assay. FIG. 5Adepicts exemplary experimental results demonstrating the lag timerequired for monomers to self-associate in the presence of pan-amyloidantibodies.

FIG. 6 depicts crystal violet assays of S. typhimurium (STM) and E. coliUTI89. STM or UTI89 were grown in the absence or presence of 3H3 (250ug/ml, 125 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ml, 1 ug/ml) as well as 0.5mg/ml control A6 or anti-Dengue antibody for 72 hours at 26 C in asterile 96 well plate. S. typhimurium curli mutant (csgBA) and UTI89curli mutant (csgBA) were also grown as negative controls.

FIG. 7 depicts exemplary experimental results demonstrating synergismbetween ALZ.3H3 and antibiotic treatment reduces S. typhimurium biofilmformation in vitro.

FIG. 8 depicts the colony forming units per biofilm grown with orwithout 3H3 and Ampicillin. Biofilms of S. typhimurium (STM) or E. coliUTI89 grown in the absence or presence of 3H3 (250 ug/ml, 125 ug/ml, 50ug/ml, 25 ug/ml, 10 ug/ml, 1 ug/ml) as well as grown with 0.5 mg/mlcontrol A6 antibody on top of sterile glass coverslips for 72 hours at26 C in a sterile 24 well dish. S. typhimurium curli mutant (csgBA) andUTI89 curli mutant (csgBA) were also grown as a negative controls. Inselect conditions, biofilms were incubated for an additional 24 hours at26 C with 30 ug/ml Ampicillin (Amp). The sterile glass coverslip, whichwas used as a surface for the biofilm to grow upon, was placed in asterile 15 ml conical tube that contained 3 ml of sterile PBS. Thebiofilm was sonicated for 10 seconds at a setting of 4 using aThermoFisher Sonic Dismembrator. Bacteria were enumerated by serialdiluting 1:10 in sterile PBS and spot plating on agar plates.

FIG. 9 depicts exemplary experimental results demonstrating that ALZ.3H3treatment reduces S. typhimurium biofilm formation in an in vitrocatheter assay.

FIG. 10 depicts exemplary experimental results demonstrating thatALZ.3H3 treatment reduces S. typhimurium biofilm formation in an in vivocatheter assay, and synergism between ALZ.3H3 and antibiotic treatmentfurther reduces S. typhimurium biofilm formation.

FIG. 11 depicts the percent survival of mice upon S. typhimuriumcatheter insertion with ALZ.3H3 and Ampicillin treatment.

FIG. 12 depicts a coomassie stained SDS:PAGE gel of amyloid beta deriveddiffusible ligands (ADDLs), also known as globulomers, produced fromAβ42.

FIG. 13 depicts the binding of anti-amyloid antibodies to Aβ42 ADDLs andAβ42 monomers, assessed by ELISA.

FIG. 14 depicts a surface plasma resonance (SPR) analysis for binding toaggregated islet amyloid peptide IAPP and tau paired helical filaments(TAU PHF) isolated from AD brain homogenates. The antibodyconcentrations used in this study were 110 nM for the IAPP and 66.7 nMfor the Tau-PHF.

FIG. 15 depicts exemplary images of binding of the 4G1 and 3H3 mAbs totau40 HEK-293 (tau40) and HEK-293 cell lines (293). Images are shownwith human mAb only (left column), TAU-5 only (center column), ormerged. 60X.

FIG. 16 depicts exemplary experimental results demonstrating thefibrillization of Aβ42 of amyloid beta oligomers alone or in presence ofanti-amyloid mAbs, 3H3, 4G1 and 4A6.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery thatantibodies having pan-amyloid binding activity have the ability toinhibit biofilm formation by preventing amyloid fibrillization.Therefore, the invention provides compositions and methods to preventbiofilm formation by targeting the amyloid component of biofilms usingpan-amyloid antibodies. In one embodiment, the invention providescompositions and methods of using an anti-amyloid monoclonal antibodythat targets biofilm by way of targeting curli. In one aspect, theinvention relates to the use of an anti-amyloid antibody wherein theantibody is specific for an amyloid beta sheet structure. In oneembodiment, the antibody is ALZ.3H3 or ALZ.4G1.

In one embodiment, the invention provides compositions and methods totarget a novel antigen such as curli and thereby change the paradigm fortreating deep-seated and foreign-body/biofilm-associated infections. Inone embodiment, the invention provides improvements to the treatment ofmulti-drug resistant bacteria by targeting an antigen that is non-crossresistant with existing drug resistance phenotypes.

In one embodiment, the invention provides a novel therapeutic targetthat allows the treatment of biofilm-producing bacteria using a humanmonoclonal antibody that can combat a diversity of gram positive andgram negative bacterial biofilms. In one embodiment, the antibody canremove a biofilm from an indwelling medical device.

In one embodiment, the invention relates to a method of treating asubject infected with a microbial species, the method comprisingadministering to the subject at least one antibody, wherein the at leastone antibody specifically binds to an amyloid beta sheet structure. Inone embodiment, the method further comprises the administration of anantibiotic. In one embodiment, the antibody of the invention (e.g.,anti-amyloid monoclonal antibody) and be combined with antibiotics toreduce the biofilm mass by targeting curli.

In another aspect, the invention relates to a method of treating,reducing, or preventing biofilm formation, wherein the compositioncomprises at least one antibody, wherein the at least one antibodyspecifically binds to an amyloid beta sheet structure. In oneembodiment, the method further comprises the administration of anantibiotic.

In one embodiment, the invention provides methods of coating the surfaceof a medical device (e.g., catheter) with a composition of the inventionto reduce biofilm mass.

In one embodiment, the invention also provides the use of thecompositions of the invention in combination with agents (such as DNase)that target other components of the extracellular matrix. In oneembodiment, the compositions of the invention can be used to treatbiofilm matrix associated with excess DNA such as Pseudomonas aeruginasabiofilms.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa.

As used herein, each of the following terms has the meaning associatedwith it in this section. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “analog” or “functional analog” refers to a related modifiedform of a polypeptide, wherein at least one amino acid substitution,deletion, or addition has been made such that said analog retainssubstantially the same biological activity as the unmodified form, invivo and/or in vitro.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.The antibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)2, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample.

As used herein, the term “antimicrobial” refers to an ability to kill orinhibit the growth of microorganisms, including but not limited tobacteria, viruses, yeast, fungi, and protozoa, or to attenuate theseverity of a microbial infection. The antimicrobial compounds orcompositions of the present invention are compounds or compositions thatmay be used for cleaning or sterilization, or may be used in thetreatment of disease and infection. The applications may include both invitro and in vivo antimicrobial uses. “Applying” an antimicrobialcomposition may include administrating a composition into a human oranimal subject.

The term “agent” includes any substance, metabolite, molecule, element,compound, or a combination thereof. It includes, but is not limited to,e.g., protein, oligopeptide, small organic molecule, glycan,polysaccharide, polynucleotide, and the like. It can be a naturalproduct, a synthetic compound, a chemical compound, or a combination oftwo or more substances. Unless otherwise specified, the terms “agent,”“substance,” and “compound” can be used interchangeably. Further, a“test agent” or “candidate agent” is generally a subject agent for usein an assay of the invention.

The term “binding” refers to a direct association between at least twomolecules, due to, for example, covalent, electrostatic, hydrophobic,ionic and/or hydrogen-bond interactions.

As used herein, the term “biofilm” refers to matrix-enclosed microbialaccretions to biological or non-biological surfaces in whichmicroorganisms are dispersed and/or form colonies. The biofilm typicallyis made of polysaccharides and other macromolecules. Biofilm formationrepresents a protected mode of growth that allows cells to survive inhostile environments.

As used herein, the term “biofilm formation” is intended to include theformation, growth, and modification of the microbial colonies containedwith biofilm structures, as well as the synthesis and maintenance of apolysaccharide matrix of the biofilm structures. Also within the scopeof this term is formation of protein-based biofilms that do not secretepolysaccharide in the matrix but which comprise proteins that permitbacteria to form biofilm architecture.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the antigen binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature 42,p 877-883.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

“Contacting” refers to a process in which two or more molecules or twoor more components of the same molecule or different molecules arebrought into physical proximity such that they are able undergo aninteraction. The term “contacting” includes, but is not limited to,impregnating, compounding, mixing, integrating, coating, rubbing,painting, spraying, immersing, rolling, smearing and dipping.

As used herein, by “combination therapy” is meant that a first agent isadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual. Suchcombinations are considered to be part of a single treatment regimen orregime.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap temporally with each other.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

As used herein, the term “heavy chain antibody” or “heavy chainantibodies” comprises immunoglobulin molecules derived from camelidspecies, either by immunization with a peptide and subsequent isolationof sera, or by the cloning and expression of nucleic acid sequencesencoding such antibodies. The term “heavy chain antibody” or “heavychain antibodies” further encompasses immunoglobulin molecules isolatedfrom an animal with heavy chain disease, or prepared by the cloning andexpression of VH (variable heavy chain immunoglobulin) genes from ananimal.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared multiplied by100. For example, if 6 of 10 of the positions in two sequences arematched or homologous then the two sequences are 60% homologous. By wayof example, the DNA sequences ATTGCC and TATGGC share 50% homology.Generally, a comparison is made when two sequences are aligned to givemaximum homology.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., 1989, Queen etal., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al.,Bio/Technology, 9:421). A suitable human acceptor antibody may be oneselected from a conventional database, e.g., the KABAT database, LosAlamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanized antibodies (see for example EP-A-0239400 andEP-A-054951).

The term “immunoglobulin” or “Ig,” as used herein, is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, the term “immune response” includes T-cell mediatedand/or B-cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity, and B cell responses, e.g., antibody production. Inaddition, the term immune response includes immune responses that areindirectly affected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages. Immune cells involved in the immune response includelymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2cells); antigen presenting cells (e.g., professional antigen presentingcells such as dendritic cells, macrophages, B lymphocytes, Langerhanscells, and non-professional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmacrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, the term “microbial organism” or “microbe,” or“microbial,” or “microorganism” refers to a domain (Bacteria) ofprokaryotic round, spiral, or rod-shaped single-celled, multi-celled, oracelled microorganisms that may lack cell walls or are Gram-positive orGram-negative or alteration thereof (i.e. Mycobacterium) if they havecell walls, that are often aggregated into colonies or motile by meansof flagella, that typically live in soil, water, organic matter, or thebodies of plants and animals, that are usually autotrophic, saprophytic,or parasitic in nutrition, and that are noted for their biochemicaleffects and pathogenicity. The term is intended to encompass prokaryoticor eukaryotic cells or organisms having a microscopic size and includesbacteria, viruses, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical. In one non-limiting example, the activityof a microbial organism can be measured by calculating the log reductionin number of the microorganism.

As used herein, the term “microbial colonization” refers to theformation of compact population groups of the same type ofmicroorganism, such as the colonies that develop when a microbial cellbegins reproducing. The microbial colonization may or may not causedisease symptoms. Decolonization refers to a reduction in the number ofmicrobial organisms present. When the microbial organisms are completelydecolonized, the microbial organisms have been eradicated and arenon-detectable.

A “mutation,” as used herein, refers to a change in nucleic acid orpolypeptide sequence relative to a reference sequence (which ispreferably a naturally-occurring normal or “wild-type” sequence), andincludes translocations, deletions, insertions, and substitutions/pointmutations. A “mutant” as used herein, refers to either a nucleic acid orprotein comprising a mutation.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintradermal (i.d.) injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereof,whether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the term “salt” embraces addition salts of free acids orfree bases that are compounds useful within the invention. Suitable acidaddition salts may be prepared from an inorganic acid or from an organicacid. Examples of inorganic acids include hydrochloric, hydrobromic,hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric andtetrafluoroboronic acids. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of whichinclude formic, acetic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable base addition salts ofcompounds useful within the invention include, for example, metallicsalts including alkali metal, alkaline earth metal and transition metalsalts such as, for example, lithium, calcium, magnesium, potassium,ammonium, sodium and zinc salts. Acceptable base addition salts alsoinclude organic salts made from basic amines such as, for example,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methyl-glucamine) and procaine. All ofthese salts may be prepared by conventional means from the correspondingfree base compound by reacting, for example, the appropriate acid orbase with the corresponding free base.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “X,”the presence of a molecule containing epitope X (or free, unlabeled A),in a reaction containing labeled “X” and the antibody, will reduce theamount of labeled X bound to the antibody.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression,diminution, remission, or eradication of a disease state.

As used herein, the term “therapeutically effective amount” of acompound of the present invention refers to an amount of the compound ofthe present invention that will elicit the biological or medicalresponse of a subject, or ameliorate symptoms, slow or delay diseaseprogression, or prevent a disease, etc. In one embodiment, the termrefers to the amount that inhibits or reduces microbial colonization orinfection. In one embodiment, the term refers to the amount thatinhibits or reduces bacterial infection, or prevent or destroying theformation of bacterial biofilms. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

As used herein, the term “treating” or “treatment” of any disease ordisorder refers in one embodiment, to ameliorating the disease ordisorder (i.e., arresting or reducing the development of the disease orat least one of the clinical symptoms thereof). In another embodiment“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the patient. In yet anotherembodiment, “treating” or “treatment” refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In yet another embodiment, “treating” or“treatment” refers to preventing or delaying the onset or development orprogression of the disease or disorder. The term “treating” or“treatment” also refers to a reduction in the severity of one or moresymptoms by about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90% or about 100%.

As used herein, the term “topical administration” refers to the deliveryto a subject by contacting the formulation directly to a surface orlocalized region of the subject. The most common form of topicaldelivery is to the skin, but a composition disclosed herein can also bedirectly applied to other surfaces of the body, e.g., to the eye, amucous membrane, to surfaces of a body cavity or to an internal surface.As mentioned above, the most common topical delivery is to the skin. Theterm encompasses several routes of administration including, but notlimited to, topical and transdermal. These modes of administrationtypically include penetration of the skin's permeability barrier andefficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.

As used herein, the term “topical formulation” (synonymously, “topicalcomposition”) is used herein to refer to a pharmaceutical preparationintended for topical or local application to an afflicted region of asubject in need thereof, and includes such dosage forms as gel, cream,ointment, emulsion, suspension, solution, drops, lotion, paint, pessary,douche, suppository, troche, spray, sponge, film, or foam. Preferably,the topical formulation is in the form of a cream, a gel, or anointment.

“Variant” of the polypeptides according to the present invention may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, (ii) one in whichthere are one or more modified amino acid residues, e.g., residues thatare modified by the attachment of substituent groups, (iii) one in whichthe polypeptide is an alternative splice variant of the polypeptide ofthe present invention, (iv) fragments of the polypeptides and/or (v) onein which the polypeptide is fused with another polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification (for example, His-tag) or for detection (for example, Sv5epitope tag). The fragments include polypeptides generated viaproteolytic cleavage (including multi-site proteolysis) of an originalsequence. Variants may be post-translationally, or chemically modified.Such variants are deemed to be within the scope of those skilled in theart from the teaching herein.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention is based, in part, on the identification of anovel therapeutic target that allows the treatment of biofilm-producingbacteria using a human monoclonal antibody which can combat a diversityof gram positive and gram negative bacterial biofilms, and even remove abiofilm from an indwelling medical device. This invention enables novelmAb drugs that target a novel antigen and will (1) change the paradigmfor treating deep-seated and foreign-body/biofilm-associated infections,(2) improve treatment of multi-drug resistant bacteria by targeting anantigen that is non-cross resistant with existing drug resistancephenotypes, and (3) be effective against a wide variety of bacteria.

The present invention provides a composition having enhancedantimicrobial efficacy and effective for inhibiting, reducing ortreating microbial infections such as bacterial infections, and/or fordecolonizing a microbial organism and/or for destroying, disrupting,inhibiting or reducing bacterial biofilm formation. Described herein isthe surprising and unexpected discovery that antibodies havingpan-amyloid binding activity disrupt biofilms from multiple microbialspecies. Further, a combination of an anti-amyloid antibody of theinvention and an antibiotic treatment, when used to treat a microbialorganism, demonstrates synergistic effect against a microbial,colonization or infection or biofilm formation. As used herein, the term“synergistic” refers to the effect obtained by combining compoundsand/or agents that is greater than the effect obtained by the separateaddition of each compound. The combination treatment of the presentinvention has shown a synergistic effect as measured by, for example,the extent of the response, the duration of response, the response rate,the stabilization rate, the duration of stabilization, the time toreduce or clear the infections, the time to eradicate themicroorganisms, to that achievable on dosing one or other of thecomponents of the combination treatment at its conventional dose. Forexample, the effect of the combination treatment of the presentinvention is synergistic because the combination treatment istherapeutically superior to the effect achievable with one componentalone or the additive effect of the combination components actingseparately. The superior effect can be improved reduction in drugresistance from the microbial organisms, the extent to which themicrobial organisms are eradicated and become non-detectable by thecombination treatment. Also for example, the effect of the combinationtreatment of the present invention is synergistic because it takesshorter time to kill the microorganisms and clear the infections. Alsofor example, the effect of the combination treatment of the presentinvention is synergistic because the combination treatment offersbroader spectrum of antimicrobial activities than those with onecomponent alone. Also for example, the effect of the combinationtreatment of the present invention is synergistic because one of thecomponents in the composition described in this invention is dosed atits conventional dose and the other component(s) is/are dosed at areduced dose and the therapeutic effect, as measured by, for example,the extent of the killing and/or inhibiting growth of the microorganismssuch as bacteria, the time to kill and/or inhibit growth of themicroorganisms such as bacteria, or the time to destroy or inhibitmicrobial colonies, or the time to disrupt or inhibit or reduce biofilmformation or growth, is equivalent to that achievable on dosingconventional amounts of the components of the combination treatment.

Compositions

The composition of the invention can treat, prevent and/or protectagainst any disease, disorder, or condition associated with a bacterialactivity. In certain embodiments, the composition can treat, prevent,and or/protect against bacterial infection. In certain embodiments, thecomposition can treat, prevent, and or/protect against bacterial biofilmformation. In certain embodiments, the invention provides a noveltherapeutic target that allows the treatment of biofilm-producingbacteria using a human monoclonal antibody that can combat a diversityof gram positive and gram negative bacterial biofilms.

In certain embodiments, the composition can treat, prevent, andor/protect against Enterobacteriaceae, Bacteroidetes, Proteobacteria,Firmicutes or Thermodesulfobacteria infection. In certain embodiments,the composition can treat, prevent, and or/protect againstEnterobacteriaceae, Bacteroidetes, Proteobacteria, Firmicutes orThermodesulfobacteria biofilm formation. In certain embodiments, thecomposition can treat, prevent, and or/protect against Salmonellatyphimurium (S typhimurium) infection. In certain embodiments, thecomposition can treat, prevent, and or/protect against S typhimuriumbiofilm formation. In certain embodiments, the composition can treat,prevent, and or/protect against Escherichia coli (E coli) infection. Incertain embodiments, the composition can treat, prevent, and or/protectagainst E coli biofilm formation. In certain embodiments, thecomposition can treat, prevent, and or/protect against Yersinia pestis(Y. pestis) infection. In certain embodiments, the composition cantreat, prevent, and or/protect against Y. pestis biofilm formation. Incertain embodiments, the composition can treat, prevent, and or/protectagainst a disease including, but not limited to, meningitis, enteritis,plague, and sepsis.

In various embodiments, the present invention includes amyloid inhibitorcompositions. In various embodiments, the amyloid inhibitor compositionsof the invention diminish or inhibit amyloid fibrillization. In oneembodiment, the amyloid inhibitor targets the major constituent ofenteric biofilms, amyloid curli. In one embodiment, the inventionprovides an amyloid inhibitor that targets curli within biofilms thatresults in alterations in biofilm architecture, stability and overallresult in reduction of the biofilm.

In one embodiment, the invention provides amyloid inhibitors that bindto and inhibit the fibrillization of bacterial amyloid curli implicatedin biofilm formation. In one embodiment, the amyloid inhibitors arehuman monoclonal antibodies that exhibit reactivity against amyloid-β aswell exhibit anti-curli properties within the context of bacterialbiofilms.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that a decrease in amyloid fibrillizationencompasses a decrease in amyloid expression, including transcription,translation, or both, and also encompasses promoting the degradation ofamyloid, including at the RNA level (e.g., RNAi, shRNA, etc.) and at theprotein level (e.g., degredation, etc.) The skilled artisan will alsoappreciate, once armed with the teachings of the present invention, thata decrease in amyloid fibrillization includes a decrease in a amyloidactivity (e.g., enzymatic activity, substrate binding activity, receptorbinding activity, etc.). Thus, decreasing amyloid fibrillizationincludes, but is not limited to, decreasing transcription, translation,or both, of a nucleic acid encoding amyloid; and it also includesdecreasing any activity of a amyloid polypeptide, or peptide fragmentthereof, as well. The amyloid inhibitor compositions and methods of theinvention can selectively inhibit amyloid, or can inhibit both amyloidand another molecule.

Inhibition of amyloid fibrillization can be assessed using a widevariety of methods, including those disclosed herein, as well as methodsknown in the art or to be developed in the future. That is, a person ofordinary skill in the art would appreciate, based upon the disclosureprovided herein, that decreasing the level or activity of amyloid can bereadily assessed using methods that assess the level of a nucleic acidencoding amyloid (e.g., mRNA), the level of a amyloid polypeptide, orpeptide fragment thereof, present in a biological sample, the level ofamyloid activity (e.g., enzymatic activity, substrate binding activity,receptor binding activity, etc.), or combinations thereof.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in treating or preventing adisease or disorder in a subject in need thereof, whether or not thesubject is also being treated with other medication or therapy. Further,the skilled artisan would further appreciate, based upon the teachingsprovided herein, that the disease or disorders treatable by thecompositions and methods described herein encompass any disease ordisorder where amyloid plays a role and where diminished amyloidfibrillization will promote a positive therapeutic outcome. The amyloidinhibitor compositions and methods of the invention that decrease thelevel or activity (e.g., amyloid fibrillization, etc.) of amyloid, or anamyloid fragment, include, but should not be construed as being limitedto, a chemical compound, a protein, a peptide, a peptidomemetic, anantibody, an antibody fragment, an antibody mimetic, a ribozyme, a smallmolecule chemical compound, an short hairpin RNA, RNAi, an antisensenucleic acid molecule (e.g., siRNA, miRNA, etc.), a nucleic acidencoding an antisense nucleic acid molecule, a nucleic acid sequenceencoding a protein, or combinations thereof. In some embodiments, theinhibitor is an allosteric inhibitor. One of skill in the art wouldreadily appreciate, based on the disclosure provided herein, that anamyloid inhibitor composition encompasses any chemical compound thatdecreases amyloid fibrillization. Additionally, an amyloid inhibitorcomposition encompasses a chemically modified compound, and derivatives,as is well known to one of skill in the chemical arts.

Further, one of skill in the art, when equipped with this disclosure andthe methods exemplified herein, would appreciate that a amyloidinhibitor composition includes such inhibitors as discovered in thefuture, as can be identified by well-known criteria in the art ofpharmacology, such as the physiological results of inhibition of amyloidas described in detail herein and/or as known in the art. Therefore, thepresent invention is not limited in any way to any particular amyloidinhibitor composition as exemplified or disclosed herein; rather, theinvention encompasses those inhibitor compositions that would beunderstood by the person of ordinary skill in the art to be useful asare known in the art and as are discovered in the future.

Further methods of identifying and producing amyloid inhibitorcompositions are well known to those of ordinary skill in the art,including, but not limited, obtaining an inhibitor from a naturallyoccurring source (e.g., Streptomyces sp., Pseudomonas sp., Stylotellaaurantium, etc.). Alternatively, a amyloid inhibitor can be synthesizedchemically. Further, the routineer would appreciate, based upon theteachings provided herein, that a amyloid inhibitor composition can beobtained from a recombinant organism. Compositions and methods forchemically synthesizing amyloid inhibitors and for obtaining them fromnatural sources are well known in the art and are described in the art.

One of skill in the art will appreciate that an inhibitor can beadministered as a chemical compound, a protein, a peptide, apeptidomemetic, an antibody, an antibody fragment, an antibody mimetic,a ribozyme, a small molecule chemical compound, an short hairpin RNA,RNAi, an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), anucleic acid encoding an antisense nucleic acid molecule, a nucleic acidsequence encoding a protein, a amyloid receptor, a amyloid receptorfragment, or combinations thereof. Numerous vectors and othercompositions and methods are well known for administering a protein or anucleic acid construct encoding a protein to cells or tissues.Therefore, the invention includes a method of administering a protein ora nucleic acid encoding a protein that is an inhibitor of amyloid.(Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York; Ausubel et al., 1997, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York).

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself increases the level or activity ofamyloid can serve in the compositions and methods of the presentinvention to decrease the level or activity of amyloid.

Antisense oligonucleotides are DNA or RNA molecules that arecomplementary to some portion of an RNA molecule. When present in acell, antisense oligonucleotides hybridize to an existing RNA moleculeand inhibit translation into a gene product. Inhibiting the expressionof a gene using an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931). The methods of the invention include the use of an antisenseoligonucleotide to diminish the amount of amyloid, or to diminish theamount of a molecule that causes an increase in the amount or activityof amyloid, thereby decreasing the amount or activity of amyloid.

Contemplated in the present invention are antisense oligonucleotidesthat are synthesized and provided to the cell by way of methods wellknown to those of ordinary skill in the art. As an example, an antisenseoligonucleotide can be synthesized to be between about 10 and about 100,more preferably between about 15 and about 50 nucleotides long. Thesynthesis of nucleic acid molecules is well known in the art, as is thesynthesis of modified antisense oligonucleotides to improve biologicalactivity in comparison to unmodified antisense oligonucleotides (Tullis,1991, U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing amyloid, or of a geneexpressing a protein that increases the level or activity of amyloid,can be accomplished through the use of a ribozyme. Using ribozymes forinhibiting gene expression is well known to those of skill in the art(see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al.,1989, Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).Ribozymes are catalytic RNA molecules with the ability to cleave othersingle-stranded RNA molecules. Ribozymes are known to be sequencespecific, and can therefore be modified to recognize a specificnucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowingthe selective cleavage of specific mRNA molecules. Given the nucleotidesequence of the molecule, one of ordinary skill in the art couldsynthesize an antisense oligonucleotide or ribozyme without undueexperimentation, provided with the disclosure and referencesincorporated herein.

Alternatively, inhibition of a gene expressing amyloid, or of a geneexpressing a protein that increases the level or activity of amyloid,can be accomplished through the use of a short hairpin RNA or antisenseRNA, including siRNA, miRNA, and RNAi. Given the nucleotide sequence ofthe molecule, one of ordinary skill in the art could synthesize such anshort hairpin RNA or antisense RNA without undue experimentation,provided with the disclosure and references incorporated herein.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder that isalready established. Particularly, the disease or disorder need not havemanifested to the point of detriment to the subject; indeed, the diseaseor disorder need not be detected in a subject before treatment isadministered. That is, significant disease or disorder does not have tooccur before the present invention may provide benefit. Therefore, thepresent invention includes a method for preventing a disease or disorderin a subject, in that an amyloid inhibitor composition, as discussedelsewhere herein, can be administered to a subject prior to the onset ofthe disease or disorder, thereby preventing the disease or disorder fromdeveloping.

The invention provides compositions that bind to amyloid. In oneembodiment, the amyloid binding agent inhibits amyloid levels oractivity. Thus, in diseases and conditions where a reduction of amyloidactivity would be beneficial, such inhibitory amyloid binding agents canpotentially act as therapeutics.

Anti-Amyloid Antibodies

Antibodies, including amyloid binding fragments thereof, of the presentinvention include, in certain embodiments, antibody amino acid sequencesdisclosed herein encoded by any suitable polynucleotide, or any isolatedor formulated antibody. Further, antibodies of the present disclosurecomprise antibodies having the structural and/or functional features ofanti-amyloid antibodies described herein. In one embodiment, theanti-amyloid antibody binds amyloid and, thereby partially orsubstantially alters at least one biological activity of amyloid (e.g.,amyloid fibrillization). In some embodiments, the amyloid is a microbialamyloid.

In one embodiment, anti-amyloid antibodies of the inventionimmunospecifically bind at least one specified epitope specific to theamyloid protein, peptide, subunit, fragment, portion or any combinationthereof and do not specifically bind to other polypeptides, other thanamyloid from other species. The at least one epitope can comprise atleast one antibody binding region that comprises at least one portion ofthe amyloid protein. The term “epitope” as used herein refers to aprotein determinant capable of binding to an antibody. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents.

The invention provides an immunological composition comprising at leastone antibody having pan-amyloid binding activity. For example, in oneembodiment, the composition comprises an antibody or antibody fragmentthat specifically binds to a beta sheet structure of an amyloid protein.Exemplary anti-amyloid antibodies include, but are not limited to,ALZ.3H3, ALZ.2C10, ALZ.4G1, and ALZ.4A6.

However, the invention should not be construed as being limited solelyto methods and compositions including these antibodies or to theseportions of the antigens. Rather, the invention should be construed toinclude other antibodies, as that term is defined elsewhere herein, toantigens, or portions thereof. Further, the present invention should beconstrued to encompass antibodies, inter alia, bind to the specificantigens of interest, and they are able to bind the antigen present onWestern blots, in solution in enzyme linked immunoassays, influorescence activated cells sorting (FACS) assays, in magenetic-activedcell sorting (MACS) assays, and in immunofluorescence microscopy of acell transiently transfected with a nucleic acid encoding at least aportion of the antigenic protein, for example.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibody can specifically bind with anyportion of the antigen and the full-length protein can be used togenerate antibodies specific therefor. However, the present invention isnot limited to using the full-length protein as an immunogen. Rather,the present invention includes using an immunogenic portion of theprotein to produce an antibody that specifically binds with a specificantigen. That is, the invention includes immunizing an animal using animmunogenic portion, or antigenic determinant, of the antigen.

Once armed with the sequence of a specific antigen of interest and thedetailed analysis localizing the various conserved and non-conserveddomains of the protein, the skilled artisan would understand, based uponthe disclosure provided herein, how to obtain antibodies specific forthe various portions of the antigen using methods well-known in the artor to be developed.

The skilled artisan would appreciate, based upon the disclosure providedherein, that that present invention includes use of a single antibodyrecognizing a single antigenic epitope but that the invention is notlimited to use of a single antibody. Instead, the invention encompassesuse of at least one antibody where the antibodies can be directed to thesame or different antigenic protein epitopes.

Methods of making and using antibodies are well known in the art. Forexample, polyclonal antibodies useful in the present invention can begenerated by immunizing rabbits according to standard immunologicaltechniques well-known in the art (see, e.g., Harlow et al., 1988, In:Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.). Suchtechniques include immunizing an animal with a chimeric proteincomprising a portion of another protein such as a maltose bindingprotein or glutathione (GSH) tag polypeptide portion, and/or a moietysuch that the antigenic protein of interest is rendered immunogenic(e.g., an antigen of interest conjugated with keyhole limpet hemocyanin,KLH) and a portion comprising the respective antigenic protein aminoacid residues. The chimeric proteins are produced by cloning theappropriate nucleic acids encoding the marker protein into a plasmidvector suitable for this purpose, such as but not limited to, pMAL-2 orpCMX.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,N.Y.).

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well-known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein. Further, the antibody of the invention may be “humanized”using the technology described in, for example, Wright et al., and inthe references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77:755-759), and other methods of humanizing antibodieswell-known in the art or to be developed.

The present invention also includes the use of humanized antibodiesspecifically reactive with epitopes of an antigen of interest. Thehumanized antibodies of the invention have a human framework and haveone or more complementarity determining regions (CDRs) from an antibody,typically a mouse antibody, specifically reactive with an antigen ofinterest. When the antibody used in the invention is humanized, theantibody may be generated as described in Queen, et al. (U.S. Pat. No.6,180,370), Wright et al., (supra) and in the references cited therein,or in Gu et al. (1997, Thrombosis and Hematocyst 77 (4):755-759). Themethod disclosed in Queen et al. is directed in part toward designinghumanized immunoglobulins that are produced by expressing recombinantDNA segments encoding the heavy and light chain complementaritydetermining regions (CDRs) from a donor immunoglobulin capable ofbinding to a desired antigen, such as an epitope on an antigen ofinterest, attached to DNA segments encoding acceptor human frameworkregions. Generally speaking, the invention in the Queen patent hasapplicability toward the design of substantially any humanizedimmunoglobulin. Queen explains that the DNA segments will typicallyinclude an expression control DNA sequence operably linked to thehumanized immunoglobulin coding sequences, includingnaturally-associated or heterologous promoter regions. The expressioncontrol sequences can be eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells or the expressioncontrol sequences can be prokaryotic promoter systems in vectors capableof transforming or transfecting prokaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the introducednucleotide sequences and as desired the collection and purification ofthe humanized light chains, heavy chains, light/heavy chain dimers orintact antibodies, binding fragments or other immunoglobulin forms mayfollow (Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NewYork, (1979), which is incorporated herein by reference).

The invention also includes functional equivalents of the antibodiesdescribed herein. Functional equivalents have binding characteristicscomparable to those of the antibodies, and include, for example,hybridized and single chain antibodies, as well as fragments thereof.Methods of producing such functional equivalents are disclosed in PCTApplication WO 93/21319 and PCT Application WO 89/09622.

Functional equivalents include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies. “Substantially the same” aminoacid sequence is defined herein as a sequence with at least 70%,preferably at least about 80%, more preferably at least about 90%, evenmore preferably at least about 95%, and most preferably at least 99%homology to another amino acid sequence (or any integer in between 70and 99), as determined by the FASTA search method in accordance withPearson and Lipman, 1988 Proc. Nat'l. Acad. Sci. USA 85: 2444-2448.Chimeric or other hybrid antibodies have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region of a monoclonal antibody from each stablehybridoma.

Single chain antibodies (scFv) or Fv fragments are polypeptides thatconsist of the variable region of the heavy chain of the antibody linkedto the variable region of the light chain, with or without aninterconnecting linker. Thus, the Fv comprises an antibody combiningsite.

Functional equivalents of the antibodies of the invention furtherinclude fragments of antibodies that have the same, or substantially thesame, binding characteristics to those of the whole antibody. Suchfragments may contain one or both Fab fragments or the F(ab′)2 fragment.The antibody fragments contain all six complement determining regions ofthe whole antibody, although fragments containing fewer than all of suchregions, such as three, four or five complement determining regions, arealso functional. The functional equivalents are members of the IgGimmunoglobulin class and subclasses thereof, but may be or may combinewith any one of the following immunoglobulin classes: IgM, IgA, IgD, orIgE, and subclasses thereof. Heavy chains of various subclasses, such asthe IgG subclasses, are responsible for different effector functions andthus, by choosing the desired heavy chain constant region, hybridantibodies with desired effector function are produced. Exemplaryconstant regions are gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), andgamma 4 (IgG4). The light chain constant region can be of the kappa orlambda type.

The immunoglobulins of the present invention can be monovalent, divalentor polyvalent. Monovalent immunoglobulins are dimers (HL) formed of ahybrid heavy chain associated through disulfide bridges with a hybridlight chain. Divalent immunoglobulins are tetramers (H2L2) formed of twodimers associated through at least one disulfide bridge.

The antibody may comprise a heavy chain and a light chaincomplementarity determining region (“CDR”) set, respectively interposedbetween a heavy chain and a light chain framework (“FR”) set whichprovide support to the CDRs and define the spatial relationship of theCDRs relative to each other. The CDR set may contain three hypervariableregions of a heavy or light chain V region. Proceeding from theN-terminus of a heavy or light chain, these regions are denoted as“CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site,therefore, may include six CDRs, comprising the CDR set from each of aheavy and a light chain V region.

The antibody may have a half-life within the subject. In someembodiments, the antibody may be modified to extend or shorten itshalf-life within the subject. The modification may be present in aconstant region of the antibody. The modification may be one or moreamino acid substitutions in a constant region of the antibody thatextend the half-life of the antibody as compared to a half-life of anantibody not containing the one or more amino acid substitutions. Themodification may be one or more amino acid substitutions in the CH2domain of the antibody that extend the half-life of the antibody ascompared to a half-life of an antibody not containing the one or moreamino acid substitutions.

In some embodiments, the one or more amino acid substitutions in theconstant region may include replacing a methionine residue in theconstant region with a tyrosine residue, a serine residue in theconstant region with a threonine residue, a threonine residue in theconstant region with a glutamate residue, or any combination thereof,thereby extending the half-life of the antibody.

In other embodiments, the one or more amino acid substitutions in theconstant region may include replacing a methionine residue in the CH2domain with a tyrosine residue, a serine residue in the CH2 domain witha threonine residue, a threonine residue in the CH2 domain with aglutamate residue, or any combination thereof, thereby extending thehalf-life of the antibody.

The antibody can be defucosylated. Fucosylation includes the addition ofthe sugar fucose to a molecule, for example, the attachment of fucose toN-glycans, O-glycans and glycolipids. Accordingly, in a defucosylatedantibody, fucose is not attached to the carbohydrate chains of theconstant region. The antibody may be modified so as to prevent orinhibit fucosylation of the antibody. The modification may be in theheavy chain, light chain, or a combination thereof. The modification maybe one or more amino acid substitutions in the heavy chain, one or moreamino acid substitutions in the light chain, or a combination thereof.

The anti-amyloid antibody can treat, prevent, and/or protect againstdisease in the subject administered the composition. The anti-amyloidantibody can promote survival of the disease in the subject administeredthe composition. In one embodiment, the anti-amyloid antibody canprovide increased survival of the disease in the subject over theexpected survival of a subject having the disease who has not beenadministered the anti-amyloid antibody. In various embodiments, theanti-amyloid antibody can provide at least about a 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or a 100% increase in survival of thedisease in subjects administered the composition over the expectedsurvival in the absence of the composition.

In one embodiment, the anti-amyloid antibody can provide increasedprotection against the disease in the subject over the expectedprotection of a subject who has not been administered the anti-amyloidantibody. In various embodiments, the anti-amyloid antibody can protectagainst disease in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% of subjects administered the compositionover the expected protection in the absence of the composition.

Combination with Antibiotic Treatment

In one aspect, the present invention provides a composition comprisingan anti-amyloid antibody of the invention in combination with at leastone additional agent. In one embodiment, the additional agent is anantibiotic. In one aspect, the present invention provides a compositioncomprising an anti-amyloid antibody of the invention and an antibiotictreatment. In one embodiment, the weight ratio between an anti-amyloidantibody of the invention and an antibiotic treatment is from about 10:1to about 1:10. In one embodiment, the weight ratio between ananti-amyloid antibody of the invention and an antibiotic treatment isfrom about 4:1 to about 1:4. In one embodiment, the weight ratio betweenan anti-amyloid antibody of the invention and an antibiotic treatment isfrom about 2:1 to about 1:2. In one embodiment, the weight ratio betweenan anti-amyloid antibody of the invention and an antibiotic treatment isabout 1:1, about 1: 2, about 1:3, about 1:4, about 1:5, about 2:1, about3:1, about 4:1, or about 5:1. In one embodiment, the total concentrationof an anti-amyloid antibody of the invention and an antibiotic treatmentin the composition of the present invention is from about 1 wt. % toabout 50 wt. %. In one embodiment, the total concentration of ananti-amyloid antibody of the invention and an antibiotic treatment inthe composition of the present invention is about 50 weight percentage(wt. %), about 40 wt. %, about 30wt. %, about 25 wt. %, about 20 wt. %,about 15 wt. %, about 10 wt. %, about 5 wt. %, about 3 wt. %, about 2wt. %, about 1 wt. % per unit of the composition.

Pharmaceutical Compositions and Formulations

The invention also encompasses a pharmaceutical composition comprisingan antibody having pan-amyloid activity. In one embodiment, thepharmaceutical composition is useful for inhibiting bacterialinfections. In one embodiment, the pharmaceutical composition is usefulfor overcoming antibacterial resistance. Such a pharmaceuticalcomposition may consist of an antibody having pan-amyloid activity in aform suitable for administration to a subject. In one embodiment, thecomposition of the invention may comprise a nucleic acid moleculeencoding an antibody having pan-amyloid activity.

In an embodiment, the pharmaceutical compositions useful for practicingthe method of the invention may be administered to deliver a dose ofbetween 1 ng/kg/day and 100 mg/kg/day. In another embodiment, thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for oral, rectal, vaginal, topical,transdermal, ophthalmic, intrathecal or another route of administration.The route(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include bringing theactive ingredient into association with a carrier or one or more otheraccessory ingredients, and then, if necessary or desirable, shaping orpackaging the product into a desired single- or multi-dose unit.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage. The unit dosage form may be for a singledaily dose or one of multiple daily doses (e.g., about 1 to 4 or moretimes per day). When multiple daily doses are used, the unit dosage formmay be the same or different for each dose.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of an antibody havingpan-amyloid activity and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers, which are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention or reduction of the action of microorganisms maybe achieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars, sodium chloride, or polyalcohols such as mannitoland sorbitol, in the composition. Prolonged absorption of the injectablecompositions may be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate orgelatin.

Formulations may be employed in admixtures with conventional excipients.The pharmaceutical preparations may be sterilized and if desired mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressurebuffers, coloring, flavoring and/or aromatic substances and the like.They may also be combined where desired with other active agents, e.g.,other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; antiseptics; antiviral agents; anticoagulants; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed, for example in Genaro, ed. (1985, Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.), which is incorporatedherein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an antioxidant and a chelating agentwhich inhibit the degradation of the compound. Preferred antioxidantsfor some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid inthe preferred range of about 0.01% to 0.3% and more preferably BHT inthe range of 0.03% to 0.1% by weight by total weight of the composition.Preferably, the chelating agent is present in an amount of from 0.01% to0.5% by weight by total weight of the composition. Particularlypreferred chelating agents include edetate salts (e.g. disodium edetate)and citric acid in the weight range of about 0.01% to 0.20% and morepreferably in the range of 0.02% to 0.10% by weight by total weight ofthe composition. The chelating agent is useful for chelating metal ionsin the composition which may be detrimental to the shelf life of theformulation. While BHT and disodium edetate are the particularlypreferred antioxidant and chelating agent respectively for somecompounds, other suitable and equivalent antioxidants and chelatingagents may be substituted therefore as would be known to those skilledin the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl para hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

The compositions of the invention may be used in aqueous emulsions suchas latexes, water-based paints and coatings, caulks and adhesives, tapejoint compounds, mineral slurries, water-cooling systems, personal careproducts, soaps and detergents, disinfectants, cleaners, and sanitizers,pesticide products, oilfield water and water-based fluids used inoilfield applications including drilling muds, fracturing fluids, andhydrotest fluids, and the like. In one embodiment, the composition is anantimicrobial composition. In one embodiment, the composition is anantiseptic.

The compositions useful within the invention may further comprise atleast one additional antimicrobial agent. Non-limiting examples of theat least one additional antimicrobial agent are levofloxacin,doxycycline, neomycin, clindamycin, minocycline, gentamycin, rifampin,chlorhexidine, chloroxylenol, methylisothizolone, thymol, α-terpineol,cetylpyridinium chloride, hexachlorophene, triclosan, nitrofurantoin,erythromycin, nafcillin, cefazolin, imipenem, astreonam, gentamicin,sulfamethoxazole, vancomycin, ciprofloxacin, trimethoprim, rifampin,metronidazole, clindamycin, teicoplanin, mupirocin, azithromycin,clarithromycin, ofoxacin, lomefloxacin, norfloxacin, nalidixic acid,sparfloxacin, pefloxacin, amifloxacin, gatifloxacin, moxifloxacin,gemifloxacin, enoxacin, fleroxacin, minocycline, linexolid,temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid,amphotericin B, fluconazole, itraconazole, ketoconazole, nystatin,penicillins, cephalosporins, carbepenems, beta-lactams antibiotics,aminoglycosides, macrolides, lincosamides, glycopeptides, tetracylines,chloramphenicol, quinolones, fucidines, sulfonamides, trimethoprims,rifamycins, oxalines, streptogramins, lipopeptides, ketolides, polyenes,azoles, echinocandines, and any combination thereof.

In one embodiment, the compound of the invention and the at least oneadditional antimicrobial agent act synergistically in preventing,reducing or treating bacterial infections. A synergistic effect may becalculated, for example, using suitable methods such as, for example,the Sigmoid-Emax equation (Holford & Scheiner, 19981, Clin.Pharmacokinet. 6: 429-453), the equation of Loewe additivity (Loewe &Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:

313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

The compositions of the invention can be formulated as appropriate fortopical administration, Accordingly the present invention provides theuse of a topical formulation for treating a microbial infection, or fordecolonizing a microbial organism, or for destroying or disrupting orinhibiting or reducing biofilm formation of a microbial organism, saidtopical formulation comprises an anti-amyloid antibody of the invention.

In one embodiment, the topical formulation of the present invention maytake the form of a cream, a lotion, an ointment, a hydrogel, a colloid,a gel, a foam, an oil, a milk, a suspension, a wipe, a sponge, asolution, an emulsion, a paste, a patch, a pledget, a swab, a dressing,a spray or a pad.

The topical formulation of the present invention comprises one or morepharmaceutically acceptable carrier. Examples of the pharmaceuticallyacceptable carriers that are usable in the context of the presentinvention include carrier materials such as a solvent, a stabilizer, asolubilizer, a filler, a tonicity enhancing agent, a structure-formingagent, a suspending agent, a dispersing agent, a chelating agent, anemulsifying agent, an anti-foaming agent, an ointment base, anemollient, a skin protecting agent, a gel-forming agent, a thickeningagent, a pH adjusting agent, a preservative, a penetration enhancer, acomplexing agent, a lubricant, a demulcent, a viscosity enhancer, abioadhesive polymer, or a combination thereof.

Examples of solvents are water or purified water, alcohols (e.g.,ethanol, benzyl alcohol), vegetable, marine and mineral oils,polyethylene glycols, propylene glycols, glycerol, and liquidpolyalkylsiloxanes.

Inert diluents or fillers may be sucrose, sorbitol, sugar, mannitol,microcrystalline cellulose, starches, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate.

Examples of buffering agents include citric acid, acetic acid, lacticacid, hydrogenophosphoric acid, diethylamine, sodium hydroxide andtromethane (/.e., tris(hydroxymethyl)aminomethane hydrochloride).

Suitable suspending agents are, for example, naturally occurring gums(e.g., acacia, arabic, xanthan, and tragacanth gum), celluloses (e.g.,carboxymethyl-, hydroxyethyl-, hydroxypropyl-, andhydroxypropylmethyl-cellulose), alginates and chitosans.

Examples of dispersing or wetting agents are naturally occurringphosphatides (e.g., lecithin or soybean lecithin), condensation productsof ethylene oxide with fatty acids or with long chain aliphatic alcohols(e.g., polyoxyethylene stearate, polyoxyethylene sorbitol monooleate,and polyoxyethylene sorbitan monooleate).

Preservatives may be added to a topical composition of the invention toprevent microbial contamination that can affect the stability of theformulation and/or cause infection in the patient. Suitable examples ofpreservatives include parabens (such as methyl, ethyl, propyl,/p-hydroxybenzoate, butyl, isobutyl, and isopropylparaben), potassiumsorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol,bronopol, bronidox, MDM hydantoin, iodopropynyl butylcarbamate,benzalconium chloride, cetrimide, and benzylalcohol.

Examples of chelating agents include sodium EDTA and citric acid.

Examples of gel bases or viscosity-increasing agents are liquidparaffin, polyethylene, fatty oils, colloidal silica or aluminum,glycerol, propylene glycol, propylene carbonate, carboxyvinyl polymers,magnesium-aluminum silicates, hydrophilic polymers (such as, forexample, starch or cellulose derivatives), water-swellablehydrocolloids, carragenans, hyaluronates, alginates, and acrylates.

Ointment bases suitable for use in the compositions of the presentinvention may be hydrophobic or hydrophilic. Ointment bases include, butare not limited to, paraffin, lanolin, liquid polyalkylsiloxanes,cetanol, cetyl palmitate, vegetal oils, sorbitan esters of fatty acids,polyethylene glycols, and condensation products between sorbitan estersof fatty acids, ethylene oxide (e.g., polyoxyethylene sorbitanmonooleate), polysorbates, white petrolatum and white wax.

Examples of humectants include, but are not limited to, ethanol,isopropanol glycerin, propylene glycol, sorbitol, lactic acid, and urea.Suitable emollients include cholesterol and glycerol.

Examples of skin protectants include, but are not limited to, vitamin E,allatoin, glycerin, zinc oxide, vitamins, and sunscreen agents.

Thickening agents are generally used to increase viscosity and improvebioadhesive properties of pharmaceutical or cosmetic compositions.Examples of thickening agents include, but are not limited to,celluloses, polyethylene glycol, polyethylene oxide, naturally occurringgums, gelatin, karaya, pectin, alginic acid, povidone, and Carbopol®polymers. Particularly interesting are thickening agents withthixotropic properties (i.e., agents whose viscosity is decreased byshaking or stirring). The presence of such an agent in a compositionallows the viscosity of the composition to be reduced at the time ofadministration to facilitate its application to the skin and, toincrease after application so that the composition remains at the siteof administration.

Bioadhesive polymers are useful to hydrate the skin and enhance itspermeability. Bioadhesive polymers can also function as thickeningagents. Examples of bioadhesive polymers include, but are not limitedto, pectin, alginic acid, chitosan, polysorbates, poly(ethyleneglycol),oligosaccharides and polysaccharides, cellulose esters and celluloseethers, and modified cellulose polymers.

Permeation enhancing agents are vehicles containing specific agents thataffect the delivery of active components through the skin. Permeationenhancing agents are generally divided into two classes: solvents andsurface active compounds (amphiphilic molecules). Examples of solventpermeation enhancing agents include, but are not limited to, alcohols(e.g., ethyl alcohol, isopropyl alcohol), dimethyl formamide, dimethylacetamide, dimethyl sulfoxide, 1-dodecylazocyloheptan-2-one,N-decyl-methylsulfoxide, lactic acid, N,N-diethyl-m-toluamide, N-methylpyrrolidone, nonane, oleic acid, petrolatum, polyethylene glycol,propylene glycol, salicylic acid, urea, terpenes, and trichloroethanol.Surfactant permeation enhancing agents may be nonionic, amphoteric,cationic, or zwitterionic. Suitable nonioinic surfactants include, butare not limited to, poly(oxyethylene)-poly(oxypropylene) blockcopolymers, commercially known as poloxamers; ethoxylated hydrogenatedcastor oils; polysorbates, such as Tween 20 or Tween 80. Amphotericsurfactants include quaternized imidazole derivatives, cationicsurfactants include cetypyridinium chloride, and zwitterionicsurfactants include the betaines and sulfobetaines. Other examples ofsuitable permeation enhancers include pentadecalactone, 2-pyrrolidine,l-dodecal-azacycloheptane-2-one, calcium thioglycolate, hexanol,derivatives of 1,3-dioxanes (i.e., 1,3-dioxacyclohexanes) and1,3-dioxalanes (i.e., 1,3-dioxacyclopentanes),l-N-dodecyl-2-pyrrolidone-5-carboxylic acid,2-pentyl-2-oxo-pyrrolidineacetic acid,2-dodecyl-2-oxo-1-pyrrolidineacetic acid, and1-azacycloheptan-2-one-2-dodecylacetic acid among others.

Medical Devices

The invention contemplates applying to or coating medical devices withthe compositions useful within the invention. Non-limiting examples ofmedical devices include disposable or permanent catheters, (e.g.,central venous catheters, dialysis catheters, long-term tunneled centralvenous catheters, short-term central venous catheters, arterialcatheters, peripherally inserted central catheters, peripheral venouscatheters, pulmonary artery Swan-Ganz catheters, urinary catheters, andperitoneal catheters, drainage catheters), long-term urinary devices,tissue bonding urinary devices, vascular grafts, vascular catheterports, wound drain tubes, ventricular catheters, hydrocephalus shuntsheart valves, heart assist devices (e.g., left ventricular assistdevices), pacemaker capsules, incontinence devices, penile implants,small or temporary joint replacements, urinary dilator, cannulas,elastomers, hydrogels, surgical instruments, dental instruments, tubings(e.g., intravenous tubes, breathing tubes, dental water lines, dentaldrain tubes, and feeding tubes), fabrics, paper, indicator strips (e.g.,paper indicator strips or plastic indicator strips), adhesives (e.g.,hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives),bandages, orthopedic implants, and any other device used in the medicalfield.

Medical devices also include any device that may be inserted orimplanted into a human being or other animal, or placed at the insertionor implantation site such as the skin near the insertion or implantationsite, and that include at least one surface which is susceptible tocolonization by microorganisms and/or biofilm-embedded microorganisms.Also contemplated within the invention is any other surface that may bedesired or necessary to prevent microorganisms and/or biofilm-embeddedmicroorganisms from growing or proliferating on at least one surface ofthe medical device, or to remove or clean microorganisms and/orbiofilm-embedded microorganisms from the at least one surface of themedical device, such as the surfaces of equipment in operating rooms,emergency rooms, hospital rooms, clinics, and bathrooms. In one specificembodiment, the composition is integrated into an adhesive, such astape, thereby providing an adhesive that may prevent or reduce growth orproliferation of microorganisms and/or biofilm embedded-microorganismson at least one surface of the adhesive.

Implantable medical devices include orthopedic implants that may beinspected for contamination or infection by microorganisms and/orbiofilm-embedded microorganisms using endoscopy. Insertable medicaldevices include catheters and shunts that can be inspected withoutinvasive techniques such as endoscopy. The medical devices may be formedof any suitable metallic materials or non-metallic materials known topersons skilled in the art. Examples of metallic materials include, butare not limited to, tivanium, titanium, and stainless steel, andderivatives or combinations thereof. Examples of non-metallic materialsinclude, but are not limited to, thermoplastic or polymeric materialssuch as rubber, plastic, polyesters, polyethylene, polyurethane,silicone, Gortex® (polytetrafluoroethylene), Dacron® (polyethylenetetraphthalate), Teflon® (polytetrafluoroethylene), latex, elastomersand Dacron® sealed with gelatin, collagen or albumin, and derivatives orcombinations thereof. The medical devices include at least one surfacefor applying the biofilm-penetrating composition. In one embodiment, thebiofilm-penetrating composition is applied to the entire medical device.

Administration

The present invention provides the use of an anti-amyloid antibody ofthe invention for treating a microbial infection, or for decolonizing amicrobial organism, or for destroying or disrupting or inhibiting orreducing biofilm formation of a microbial organism.

In one embodiment, the composition described herein is foradministration to a subject. In one embodiment the subject has microbialinfection or colonization by microbes. Preferably the microbialinfection or colonization site is characterized with microbial coloniesor biofilm or biofilm formation. Preferably the microbial infection is abacterial infection. In one embodiment, the bacteria infection is fromGram-positive or Gram-negative bacteria. In one embodiment the bacterialinfection is from one selected from Staphylococcus spp., e.g.Staphylococcus aureus, Staphylococcus epidermidis; Enterococcus spp.,e.g. Enterococcus faecalis; Klebsiella spp., e.g. Klebsiella pneumoniae;Acinetobacter spp., e.g. Acinetobacter baumannii; Pseudomonas spp., e.g.Pseudomonas aeruginosa; Enterobacter spp.; Streptococcus pyogenes;Listeria spp.; Pseudomonas spp.; Mycobacterium spp., e.g. Mycobacteriumtuberculosis; Enterobacter spp.; Campylobacter spp.; Salmonella spp.;Streptococcus spp., e.g. Streptococcus Group A or B, Streptoccocuspneumoniae; Helicobacter spp., e.g. Helicobacter pylori; Neisseria spp.,e.g. Neisseria gonorrhea, Neisseria meningitidis; Borrelia burgdorferi;Shigella spp., e.g. Shigella flexneri; Escherichia coli; Haemophilusspp., e.g. Haemophilus influenzae; Chlamydia spp., e.g. Chlamydiatrachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Francisellafularensis; Bacillus spp., e.g. Bacillus anthracis; Clostridia spp.,e.g. Clostridium botulinum; Yersinia spp., e.g. Yersinia pestis;Treponema spp.; Burkholderia spp.; e.g. Burkholderia mallei and Bpseudomallei, or the combination thereof. Also in one embodiment, thebacteria are selected from Acidothermus cellulyticus, Actinomycesodontolyticus, Alkaliphilus metalliredigens, Alkaliphilus oremlandii,Arthrobacter aurescens, Bacillus amyloliquefaciens, Bacillus clausii,Bacillus halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillussubtilis, Bifidobacterium adolescentis, Bifidiobacterium longum,Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans,Clostridium acetobutylicum, Clostridium beijerinckii, Clostridiumbotulinum, Clostridium cellulolyticum, Clostridium difficile,Clostridium kluyveri, Clostridium leptum, Clostridium novyi, Clostridiumperfringens, Clostridium tetani, Clostridium thermocellum,Corynebacterium diphtherias, Corynebacterium efficiens, Corynebacteriumglutamicum, Corynebacterium jeikeium, Corynebacterium urealyticum,Desulfitobacterium hafniense, Desulfotomaculum reducens, Eubacteriumventriosum, Exiguobacterium sibiricum, Finegoldia magna, Geobacilluskaustophilus, Geobacillus thermodenitrificans, Janibacter sp.,Kineococcus radiotolerans, Lactobacillus fermentum, Listeriamonocytogenes, Listeria innocua, Listeria welshimeri, Moorellathermoacetica, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumgilvum, Mycobacterium leprae, Mycobacterium paratuberculosis,Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacteriumulcerans, Mycobacterium vanbaalenii, Nocardioides sp., Nocardiafarcinica, Oceanobacillus iheyensis, Pelotomaculum thermopropionicum,Rhodococcus sp., Saccharopolyspora erythraea, coagulase-negativeStaphylococcus species, Staphylococcus aureus, methicillin resistantStaphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillinresistant Staphylococcus epidermidis, (MRSE), Staphylococcuspseudintermedius, Staphylococcus intermedius, Staphylococcus delphini,Streptococcus agalactiae, Streptococcus gordonii, Streptococcus mitis,Streptococcus oralis, Streptococcus pneumoniae, Streptococcus sanguinis,Streptococcus suis, Streptomyces avermitilis, Streptomyces coelicolor,Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis, or thecombination thereof.

In one embodiment, the biofilm formation is on a surface of a device. Inone embodiment, the device is a medical device. In one embodiment, thebiofilm formation is on a surface of or in a tissue of a subject. In oneembodiment, the biofilm formation is on a skin, eye, a mucous membrane,surface of cavity, etc.

Accordingly the present invention provides the use of a compositioncomprising an anti-amyloid antibody of the invention for treating amicrobial infection, or for decolonizing a microbial organism, or fordestroying or disrupting or inhibiting or reducing biofilm formation ofa microbial organism. In one embodiment, the use is for decolonizing amicrobial organism, or for disrupting or inhibiting or reducing biofilmformation on a surface of a medical device.

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to or after the onset of pathogenic colonization, biofilmformation, and/or infection in a patient. Further, several divideddosages, as well as staggered dosages may be administered daily orsequentially, or the dose may be continuously infused, or may be a bolusinjection. Further, the dosages of the therapeutic formulations may beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto prevent, reduce or disrupt pathogenic colonization, biofilmformation, and/or infection in the patient. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the activity of the particular compoundemployed; the time of administration; the rate of excretion of thecompound; the duration of the treatment; other drugs, compounds ormaterials used in combination with the compound; the state of thedisease or disorder, age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell-known in the medical arts. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation. Anon-limiting example of an effective dose range for a therapeuticcompound of the invention is from about 0.01 and 50 mg/kg of bodyweight/per day. One of ordinary skill in the art would be able to studythe relevant factors and make the determination regarding the effectiveamount of the therapeutic compound without undue experimentation.

The compound can be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of breathing control disorders in a patient.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), vegetable oils,and suitable mixtures thereof. The proper fluidity may be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prevention of the action of microorganismsmay be achieved by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. In some embodiments, it is useful to include isotonic agents,for example, sugars, sodium chloride, or polyalcohols such as mannitoland sorbitol, in the composition. Prolonged absorption of the injectablecompositions can be achieved by including in the composition an agentwhich delays absorption, for example, aluminum monostearate or gelatin.In one embodiment, the pharmaceutically acceptable carrier is DMSO,alone or in combination with other carriers.

The therapeutically effective amount or dose of a compound of thepresent invention depends on the age, sex and weight of the patient, thecurrent medical condition of the patient and the severity of the diseaseor infection in the patient being treated. The skilled artisan is ableto determine appropriate doses depending on these and other factors.

The dose may be administered in a single dosage or in multiple dosages,for example from 1 to 4 or more times per day. When multiple dosages areused, the amount of each dosage may be the same or different. Forexample, a dose of 1 mg per day may be administered as two 0.5 mg doses,with about a 12-hour interval between doses.

Doses of the composition of the invention for administration may be inthe range of from about 1 μg to about 10,000 mg, from about 20 μg toabout 9,500 mg, from about 40 μg to about 9,000 mg, from about 75 μg toabout 8,500 mg, from about 150 μg to about 7,500 mg, from about 200 μgto about 7,000 mg, from about 3050 μg to about 6,000 mg, from about 500μg to about 5,000 mg, from about 750 μg to about 4,000 mg, from about 1mg to about 3,000 mg, from about 10 mg to about 2,500 mg, from about 20mg to about 2,000 mg, from about 25 mg to about 1,500 mg, from about 30mg to about 1,000 mg, from about 40 mg to about 900 mg, from about 50 mgto about 800 mg, from about 60 mg to about 750 mg, from about 70 mg toabout 600 mg, from about 80 mg to about 500 mg, and any and all whole orpartial increments therebetween.

In some embodiments, the dose of a composition of the invention is fromabout 1 mg to about 2,500 mg. In some embodiments, a dose of acomposition of the invention used in compositions described herein isless than about 10,000 mg, or less than about 8,000 mg, or less thanabout 6,000 mg, or less than about 5,000 mg, or less than about 3,000mg, or less than about 2,000 mg, or less than about 1,000 mg, or lessthan about 500 mg, or less than about 200 mg, or less than about 50 mg.Similarly, in some embodiments, the dosage of a second composition asdescribed elsewhere herein is less than about 1,000 mg, or less thanabout 800 mg, or less than about 600 mg, or less than about 500 mg, orless than about 400 mg, or less than about 300 mg, or less than about200 mg, or less than about 100 mg, or less than about 50 mg, or lessthan about 40 mg, or less than about 30 mg, or less than about 25 mg, orless than about 20 mg, or less than about 15 mg, or less than about 10mg, or less than about 5 mg, or less than about 2 mg, or less than about1 mg, or less than about 0.5 mg, and any and all whole or partialincrements thereof.

The compositions for use in the method of the invention may beformulated in unit dosage form. The term “unit dosage form” refers tophysically discrete units suitable as unitary dosage for patientsundergoing treatment, with each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

In one embodiment, the compositions of the invention are administered tothe patient from about one to about five times per day or more. Invarious embodiments, the compositions of the invention are administeredto the patient, 1-7 times per day, 1-7 times every two days, 1-7 timesevery 3 days, 1-7 times every week, 1-7 times every two weeks, and 1-7times per month. It is readily apparent to one skilled in the art thatthe frequency of administration of the various combination compositionsof the invention will vary from individual to individual depending onmany factors including, but not limited to, age, the disease or disorderto be treated, the severity of the disease or disorder to be treated,gender, overall health, and other factors. Thus, the invention shouldnot be construed to be limited to any particular dosing regime and theprecise dosage and composition to be administered to any patient isdetermined by the medical professional taking all other factors aboutthe patient into account.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's condition has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, may be reduced to a level at whichthe improved disease is retained. In some embodiments, a patient mayrequire intermittent treatment on a long-term basis, or upon anyrecurrence of the disease or disorder.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compositions lies preferablywithin a range of circulating concentrations that include the ED₅₀ withminimal toxicity. The dosage optionally varies within this rangedepending upon the dosage form employed and the route of administrationutilized.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a composition of the invention,alone or in combination with a second pharmaceutical agent; andinstructions for using the composition to treat or prevent a disease orinfection in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compositions for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Routes of Administration

Routes of administration of any of the compositions of the inventioninclude oral, rectal, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (trans)rectal, intravesical, and topical administration.

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acomposition through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments, gels,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/v) active ingredient in a solvent, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide (DMSO), and the like. Other enhancersinclude oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram,alkanecarboxylic acids, dimethylsulfoxide, polar lipids, orN-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see Constanza, U.S.Pat. No. 6,323,219).

In alternative embodiments, the topically active pharmaceuticalcomposition may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In another embodiment, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another aspect, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in anamount effective to affect desired changes. As used herein “amounteffective” shall mean an amount sufficient to cover the region of skinsurface where a change is desired. An active composition should bepresent in the amount of from about 0.0001% to about 15% by weightvolume of the composition. More preferable, it should be present in anamount from about 0.0005% to about 5% of the composition; mostpreferably, it should be present in an amount of from about 0.001% toabout 1% of the composition. Such compositions may be synthetically-ornaturally derived.

For oral administration, suitable forms include tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions formulated for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compositions of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation involves the use of materials that are solid orsemi-solid at room temperature (i.e., having a relatively low softeningor melting point range) to promote granulation of powdered or othermaterials, essentially in the absence of added water or other liquidsolvents. The low melting solids, when heated to a temperature in themelting point range, liquefy to act as a binder or granulating medium.The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.,drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compositions ofthe invention, and a further layer providing for the immediate releaseof a medication for treatment of G-protein receptor-related diseases ordisorders. Using a wax/pH-sensitive polymer mix, a gastric insolublecomposition may be obtained in which the active ingredient is entrapped,ensuring its delayed release.

For parenteral administration, the compositions of the invention may beformulated for injection or infusion, for example, intravenous,intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In one embodiment, the formulations of the present invention may be, butare not limited to, short-term, rapid-offset, as well as controlled, forexample, sustained release, delayed release and pulsatile releaseformulations.

The term sustained release refers to a drug formulation that providesfor gradual release of a drug over an extended period of time, and thatmay, although not necessarily, result in substantially constant bloodlevels of a drug over an extended time period. The period of time may beas long as a day, a week, or a month or more and should be a releasewhich is longer that the same amount of agent administered in bolusform. The term delayed release is used herein in its conventional senseto refer to a drug formulation that provides for an initial release ofthe drug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

For sustained release, the compositions may be formulated with asuitable polymer or hydrophobic material which provides sustainedrelease properties to the compositions. As such, the compositions foruse the method of the invention may be administered in the form ofmicroparticles, for example, by injection or in the form of wafers ordiscs by implantation.

In one embodiment of the invention, the compositions of the inventionare administered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term pulsatile release refers to a drug formulation that providesrelease of the drug in such a way as to produce pulsed plasma profilesof the drug after drug administration.

The term immediate release refers to a drug formulation that providesfor release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

Combination Therapy

The anti-amyloid antibody of the present invention may be administeredalone or in conjunction with another therapy. For example, thecombination therapy of the present invention may be used in conjunctionwith a disinfectant, antiseptic, antibiotic, or biocide on a surfacesuch as medical devices and indwelling devices including stents,catheters, peritoneal dialysis tubing, draining devices, jointprostheses, dental implants and the like.

In one embodiment, the present invention provides a synergisticcombination therapy comprising an anti-amyloid antibody of the inventionand an antibiotic treatment that can be administered topically for thetreatment of a microbial colonized surface or infection.

In another aspect, the present invention provides a method of treating amicrobial infection in a subject comprising administering to the subjectseparately, simultaneously or sequentially a therapeutically effectiveamount of an anti-amyloid antibody of the invention and an antibiotictreatment. In one embodiment, the method comprises administering to thesubject a therapeutically effective amount of a composition comprisingan anti-amyloid antibody of the invention and an antibiotic treatmentdescribed herein. In one embodiment, the method comprises administeringto the subject a therapeutically effective amount of a topicalformulation comprising an anti-amyloid antibody of the invention and anantibiotic treatment described herein and one or more pharmaceuticallyacceptable carriers or excipients, wherein the topical formulation andthe pharmaceutically acceptable carriers or excipients are definedherein throughout the specification. In one embodiment, the infection isa topical infection. The topical infection is an infection on a surfaceor localized region of a subject including skin, eye, a mucous membrane,a surface of cavity, etc. In one embodiment, the topical infection isthe infection on the skin. In one embodiment, the topical infection isin the form of wound, ulcer and lesion. In one embodiment, the microbialorganism is a bacterium.

In one embodiment, the present invention provides a method ofdecolonizing a microbial organism comprising contacting the microbialorganism separately, simultaneously or sequentially with an anti-amyloidantibody of the invention and an antibiotic treatment. In oneembodiment, the method comprises contacting the microbial organism witha composition comprising an anti-amyloid antibody of the invention andan antibiotic treatment described herein. In one embodiment, the methodcomprises contacting the microbial organism with a topical formulationcomprising an anti-amyloid antibody of the invention and an antibiotictreatment described herein and one or more pharmaceutically acceptablecarriers or excipients, wherein the topical formulation and thepharmaceutically acceptable carriers or excipients are defined hereinthroughout the specification. According to any of the methods describesherein, the microbial organism is a bacterium.

In one embodiment, the biofilm formation is on a surface of a device. Inone embodiment, the device is implanted catheters, prosthetic heartvalves, cardiac pacemakers, contact lenses, cerebrospinal fluid shunts,joint replacements or intravascular lines. In one embodiment, thebiofilm formation is on a surface of or in a tissue of a subject. In oneembodiment, the biofilm formation is on a skin, eye, a mucous membrane,surface of cavity, etc.

In one embodiment, the present invention provides a method of destroyingor disrupting or inhibiting or reducing biofilm formation of a microbialorganism comprising contacting the microbial organism separately,simultaneously or sequentially with a composition comprising ananti-amyloid antibody of the invention and a composition comprising anantibiotic. In one embodiment, the method comprises contacting themicrobial organism with a composition comprising an anti-amyloidantibody of the invention and an antibiotic described herein. In oneembodiment, the method comprises contacting the microbial organism witha topical formulation comprising an anti-amyloid antibody of theinvention and an antibiotic treatment described herein and one or morepharmaceutically acceptable carriers or excipients, wherein thepharmaceutically acceptable carriers or excipients are defined hereinthroughout the specification. In one embodiment, the microbial organismis a bacterium.

These methods described herein are by no means all inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compositionsknown to exert the desired effect.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: Anti-Amyloid Directed Monoclonal Antibodies Reduce BiofilmFormation of Salmonella typhimurium by Targeting Amyloid Curli

Amyloids from prokaryotic and eukaryotic origin do not share primarysequence structure, however amyloids from both lineages share a commonconserved beta sheet structure (Rapsinski et al., 2013, J Biol Chem, 17;288 (20):14178-88). Furthermore both bacterial and host amyloids bind toand activate TLR2. Similar to the binding of curli to TLR2 on innateimmune cells (Tiikel et al., 2010, Cell Microbiol, 12 (10):1495-505),binding of amyloid beta associated in Alzheimer's disease (Liu et al.,2012, J Immunol, 188 (3):1098-107; Udan et al., 2008, J Neurochem, 104(2):524-33) and Serum Amyloid A in atherosclerosis (Seidl et al., 2017,PLoS One, 12 (3):e0171711). The ability of the monoclonal antibodies tobind to and inhibit the fibrillization of bacterial amyloid curliimplicated in biofilm formation was tested. Using a multi-disciplinaryapproach, various human monoclonal antibodies were identified thatexhibit reactivity against amyloid-β that as well exhibit anti-curliproperties within the context of S. typhimurium biofilms. Incubation ofS. typhimurium (STM) biofilms with the mAb altered the biofilmarchitecture, destabilized the biofilm and ultimately reduced biomass.The resulting alterations to the biofilm architecture and stabilityrendered the biofilm more sensitive to treatment with antibiotics, DNaseand macrophage uptake of bacteria in comparison to biofilms that did notreceive mAb treatment. Overall, a novel therapeutic method has beenidentified by which targeting curli within the biofilm of S. typhimuriumbiofilms through the use of anti-amyloid mAbs results in alterations inbiofilm architecture, stability and overall result in reduction of thebiofilm.

The experimental methods and results are now described.

Monoclonal Antibody ALZ.3H3 Disrupts S. typhimurium Biofilm Architectureand Integrity

Biofilms were established in the presence of 0.5 mg/ml ALZ.3H3 for 72hours at 28° C. After growth, biofilms were stained with Syto9 andimaged using CSLM. Three dimensional architecture was examined bycreating 3D surface plots using ImageJ. Incubation of the monoclonalantibody ALZ.3H3 altered the architecture of biofilm forming a dispersebiofilm topography that expanded above the mean thickness (20 μm) of awild-type S. typhimurium biofilm which displayed a dense and compactarchitecture (FIG. 1A). This loose matrix was also observed in theanti-csg treatment, while biofilms treated with control A6 remainedintact and undisrupted (FIG. 1A). To quantify the disperse biofilmcreated by treatment with ALZ.3H3, through the use of ImageJ allparticles above the thickness of a wild-type S. typhimurium (20 μm) wasused to set the threshold value, and these threshold parameters wereapplied to subsequent treatments (FIG. 1B). Upon enumeration of theparticles, there was a significant increase in number of particles upontreatment with ALZ.3H3 (FIG. 1C). To this effect, by platingsupernatants recovered from the biofilms, significantly more bacteriawas recovered from the biofilm supernatant of ALZ.3H3. The bacteriarecovered from the supernatants is representative of the loose dispersebiofilm matrix that is easily dissociated from the biomass. Overall,this data suggests that incubation with ALZ.3H3 altered the biofilmstructure by disrupting the overall biofilm matrix structure.

Monoclonal Antibody ALZ.3H3 Alters Biofilm Integrity of Pre-EstablishedS. typhimurium Biofilms

To determine if ALZ.3H3 can reduce biofilm formation of pre-establishedbiofilms of S. typhimurium, 0.5 mg/ml of ALZ.3H3 was added for anadditional 24 hours to a 24 hour pre-established biofilm of S.typhimurium, or 0.5 mg/ml of ALZ.3H3 was added for 24 hours to a 48 hourpre-established biofilms. These biofilms were then compared,respectively, to a 48 and 72 hour S. typhimurium biofilm that was notexposed to ALZ.3H3 (FIG. 2A). Biofilms were stained with Syto9 andimaged using CSML. Biofilms were not wash extensively in order determinethe impact of ALZ.3H3 on biofilm architecture. In comparison to a 48hour biofilm of wild-type S. typhimurium, although still establishingand the biofilm matrix was incomplete, the 24 hours pre-establishedbiofilm that was exposed to ALZ.3H3 for an additional 24 hours began toexhibit a more disperse matrix in comparison to the 48 hour biofilm wascompact (FIG. 2B). This trend continued when a 48 hour pre-establishedbiofilm was incubated for an additional 24 hours with 0.5 mg/ml ALZ.3H3where the biofilm matrix was more disperse in comparison to the 72 hourfully formed S. typhimurium biofilm that did not receive ALZ.3H3treatment (FIG. 2B). Using the thresholding technique described earlier,the number of particles above the thickness of the wild-type S.typhimurium were counted. Even as early as 24 hours, incubation withALZ.3H3 increased the number of particles above the wild-type S.typhimurium, and the number of particles increased as well when a 48hour pre-established biofilm of S. typhimurium was incubated withALZ.3H3 for 24 hours (FIG. 2C). This data suggests that ALZ.3H3 impactsthe structure pre-established biofilms of S. typhimurium as early as 24hours after addition of ALZ.3H3.

Dosage Response of ALZ.3H3 on Biofilm Integrity

A crystal violet assay dose curve of 3H3 (0.1 ug/ml, 1 ug/ml, 10 ug/ml,25 ug/ml, 50 ug/ml, 250 ug/ml 500 ug/ml and untreated) added duringbiofilm growth of S. typhimurium, E. coli MC4100, S. enteritidis and E.coli UTI89 was performed and the biomass determined at absorbance at 570nm (FIG. 3).

Confocal analysis of biofilms of S. typhimurium grown in the presence(10 ug/ml, 25 ug/ml, 50 ug/ml, 250 ug/ml, 500 ug/ml) or absence(untreated) of 3H3 were performed. Biofilms were stained with syto9(green nucleic acid stain for bacteria) and amyloid dye Congo red (redcurli) and were imaged using Leica TCS confocal at 63×. Biofilm 3Dreconstructions created using ImageJ 3D viewer software (FIG. 4A).Biofilm thickness (um) of biofilms of S. typhimurium grown in thepresence (10 ug/ml, 25 ug/ml, 50 ug/ml, 250 ug/ml, 500 ug/ml) or absence(untreated) of 3H3 was measure measured using Leica TCS confocalmicroscopy software (FIG. 4C).

ALZ.3H3 Reduces Fibrillization of Curli

The formation of monomers into mature curli fibrils can be monitored byincubation with Thioflavin T (ThT). Fluorescence of ThT increases as itbinds to amyloid fibril, allowing the amyloid formation process to bemeasured spectroscopically. The formation of amyloid fibers can bedefined by a three stage sigmoidal curve (Dueholm et al., 2011,Biochemistry, 50 (39):8281-90; Wang et al., 2007, J Biol Chem, 282(6):3713-9). In the first stage, monomeric subunits are slow toself-associate and ThT fluorescence is minimal (Naiki et al., 1991, LabInvest, 65 (1):104-10). The reaction exponential increases as monomericseeds oligomerize and continue to elongate (Naiki et al., 1991, LabInvest, 65 (1):104-10). This process is marked by a rapid increase inThT fluorescence (Naiki et al., 1991, Lab Invest, 65 (1):104-10). Theoligomers elongate into mature fibrils and Tht fluorescence peaks(Dueholm et al., 2011, Biochemistry, 50 (39):8281-90; Wang et al., 2007,J Biol Chem, 282 (6):3713-9). This reaction plateaus once all monomershave been consumed and fibril elongation ceases (Naiki et al., 1991, LabInvest, 65 (1):104-10). Histadine tagged CsgA (His-CsgA), which willnaturally self-associate and fibrillize, was incubated with 0.5 mg/mlALZ.3H3 and the fibrillization reaction was monitored using ThT. Ascontrols synthetic peptides CsgAR₄₋₅ and CsgAR_(4-5N122A) were incubatedwith Tht and the fribrillization reaction was monitored in parallel.CsgAR₄₋₅ is the fourth and fifth repeat of CsgA which has been shown toself-associate and form fibrils, whereas CsgAR_(4-5N122A) contains asingle amino acid mutation that prevents fibrillization (Tükel et al.,2009, Cell Host Microbe, 6 (1):45-53). Upon fibrillization with His-CsgAand ALZ.3H3, a significant reduction in the relative fluorescence unitswas observed (FIG. 5A). In addition to determining the maximum relativefluorescent units that are representative of fibril formation, the lagtime (t₀) can be calculated using the equation t₀=t_(1/2)−2t_(e), wheret_(1/2) is the time required to reach half the maximum fluorescenceintensity and t_(e) is the elongation time (Naiki et al., 1991, LabInvest, 65 (1):104-10). The lag time can be defined as the amount oftime required for monomers to self-associate and begin to oligomerize(Naiki et al., 1991, Lab Invest, 65 (1):104-10). In comparison to thelag time calculate for the synthetic peptide CsgAR₄₋₅, there was asignificant increase in the lag times required for His-CsgA toself-associate and oligomerize when incubated with ALZ.3H3 (FIG. 5B).Based on this data, it proposed that ALZ.3H3 interacts on the monomericlevel to prevent fibrillization. By interacting with the monomer,ALZ.3H3 increases the time required for the monomers to self-associatethere by preventing fibril formation.

Crystal Violet Assay of S. typhimurium (STM) or E. coli UTI89

STM or UTI89 were grown in the absence or presence of 3H3 (250 ug/ml,125 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ml, 1 ug/ml) as well as 0.5 mg/mlcontrol A6 or anti-Dengue antibody for 72 hours at 26 C in a sterile 96well plate. S. typhimurium curli mutant (csgBA) and UTI89 curli mutant(csgBA) were also grown as negative controls. More consistent resultswith experiments involving STM, and more variability involvingexperiments with UTI89 (FIG. 6).

Synergism Between ALZ.3H3 and Antibiotic Treatment Reduces S.typhimurium Biofilm Formation

Biofilms enhance the recalcitrance of bacteria to antibiotics (Keren etal., 2004, FEMS Microbiol Lett, 230 (1):13-8, Brown et al., 1988, JAntimicrob Chemother, 22 (6):777-80, Stewart, 2002, Int J Med Microbiol,292 (2):107-13). To enhance the clearance of bacterial biofilms, thestructure of the biofilm was altered by employing ALZ.3H3 and then thebiofilm was subjected to antibiotics to kill the bacteria within thedisperse biofilm. To do this, biofilms of S. typhimurium wereestablished in the presence or absence of 0.5 mg/ml ALZ.3H3, after whichbiofilms were exposed to ampicillin for an additional 24 hours. As DNaseis an additional integral component of S. typhimurium biofilms, wildtypeor ALZ.3H3 incubated biofilms were exposed to X DNase treatment for anadditional 24 hours. Biofilms were then stained with Syto9 andvisualized using CSML. Excessive washing steps were avoided in aims toexamine the architecture of the biofilms. As antibiotic treatments arerelatively ineffective against biofilm grown, no alteration in overallbiofilm appearance was observed when wildtype biofilms were treated withampicillin or DNase following biofilm establishment. However, when S.typhimurium biofilms were incubated in the presence of ALZ.3H3 andantibiotic treatment, there was a significant reduction in the biofilmmass to the levels below detection (FIG. 7).

Biofilms of S. typhimurium (STM) or E. coli UTI89 grown in the absenceor presence of 3H3 (250 ug/ml, 125 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ml,1 ug/ml) as well as grown with 0.5 mg/ml control A6 antibody on top ofsterile glass coverslips for 72 hours at 26 C in a sterile 24 well dish.S. typhimurium curli mutant (csgBA) and UTI89 curli mutant (csgBA) werealso grown as a negative controls. In select conditions, biofilms wereincubated for an additional 24 hours at 26 C with 30 ug/ml Ampicillin(Amp). To determine the number of colony forming units per biofilm(CFU/biofilm), the sterile glass coverslip which was used as a surfacefor the biofilm to grow upon, was placed in a sterile 15 ml conical tubethat contained 3 ml of sterile PBS. The biofilm was sonicated for 10seconds at a setting of 4 using a ThermoFisher Sonic Dismembrator.Previous experiments confirmed that sonication at the previouslymentioned settings did not kill the bacteria. Bacteria were enumeratedby serial diluting 1:10 in sterile PBS and spot plating on agar plates.A reduction of CFU/biofilm was observed when STM and UTI89 are grownwith 3H3, and an additional reduction in CFU/biofilm was observed whenAmpicillin was added for an additional 24 hours (although this reductionmay be enhanced with longer Ampicillin exposure times) (FIG. 8).

To investigate the ability of ALZ.3H3 to eliminate biofilm growth in aphysiologically relevant model, biofilms of S. typhimurium were grown onmedical grade catheters in the presence or absence of ALZ.3H3 and theimpact on catheter associated biofilm architecture was investigated.Salmonella biofilms were grown alone or in the presence of ALZ3H3antibody upon sterile i.v. catheters. Bacteria were labeled green usingGFP. Congo red was used to stain for curli amyloid fibrils. When grownin the presence of 0.5 mg/ml ALZ3H3 mAbs for 72 hours, the biofilm wasdispersed. There was no significant staining with Congo red suggestingthe lack of curli fibers on the biofilm (FIG. 9). To investigate thecombination effect of ALZ3H3 and ampicillin on biofilm growth, mice weregiven 1 mg/ml Ampicillin in the drinking water 24 hours prior tocatheter insertion. Sterile i.v. catheters were incubated for 24 hourswith Salmonella typhimurium prior to insertion into the back flanks ofBalb/C mice. 24 and 28 hours after catheter insertion 100 ug of ALZ3H3was percutaneously inserted into the lumen of the catheter. 72 hoursafter catheter insertion, mice were euthanized and the catheters wereremoved. Bacteria were labeled green using GFP. Congo red was used tostain for curli amyloid fibrils. Catheters autofluoresced green.Untreated Salmonella typhimurium biofilms adhered closely to thecatheter wall with curli throughout the biomass whereas biofilms treatedwith ALZ3H3 had a disperse morphology with little curli. Combinationtreatment with ALZ3H3 and ampicillin markedly reduced the biofilm growthon the catheter which displayed little to no amyloid incorporation (FIG.10).

Catheters were colonized with biofilms of S. typhimurium (STM) and onecolonized catheter was inserted into each of 15 C67BL/6 mice (Cohorts: 5mice that received STM catheters only, 5 mice that received STMcatheters and percutaneous injections of 100 ug 3H3 and 5 mice thatreceived STM catheters, percutaneous injections of 100 ug 3H3, and 1mg/ml ampicillin (Amp) ad libitum in the drinking water. (**Ampicillinwas added to drinking water 24 hours prior to catheter insertion).Beginning 24 hours after catheter insertion, in appropriate groups 100ug/ml of 3H3 was inserted percutaneously into the lumen of the cathetersevery 24 hours post catheter insertion. Mice were monitored daily forsurvival (FIG. 11).

Example 2: Development of Pan-Amyloid Antibodies

Bacteria form multicellular communities termed biofilms in the natureand during infections to protect themselves against insults includingantimicrobials and immune system. The extracellular matrix (ECM) of abiofilm is composed of polysaccharides, DNA, and proteins, includingamyloids. Amyloids are naturally occurring, insoluble fibrillaryproteins defined by a cross-beta sheet secondary structure. Congo Red isa specific dye that binds to amyloids and can be used to identify thebacterial amyloids. Bacteria uses utilize these proteins to decoratetheir biofilms. One of the best-studied bacterial amyloids is curli,specifically produced by Enterobacteriaceae. A human monoclonal antibody(mAb) that exhibits pan-amyloid binding activity inhibits formation ofbiofilms of both E. coli and Salmonella typhimurium in vitro and invivo.

Amyloid beta derived diffusible ligands (ADDLs), also known asglobulomers, produced from Aβ42 (FIG. 12). These ADDLs were used toscreen for human mAbs that bind amyloid-specific epitopes. Hybridomaswere created from B cells of a patient with a clinical diagnosis ofmild-moderate Alzheimer's Disease, and three newly cloned mAbs (4G1,4A6, and 2C10) were compared to ALZ.3H3 (FIG. 13). Monomeric Aβ42adherent to an ELISA plate can adopt a conformation recognized by mAbsspecific for conformational amyloid epitopes (Levites et al., 2015, JNeurosci, 35 (16): 6265-6276).

MAb binding to Aβ42 conformers was measured by surface plasma resonance(SPR) (Table 1). Calculated affinity constants (K_(D)) were obtainedusing Qdat software. Compared to the the N-terminal biotinylatedmonomer, all of the mAbs exhibit lower affinity binding to some or allof the Aβ42 oligomers or fibrils. The 3H3 binding data is qualitativelymost similar to the 4G1 and 6E10 mAbs. 4A6 and 2C10 preferentially bindAβ fibrils.

TABLE 1 MAb binding to Aβ42 conformers. Oligomers (ADDLs/globulomers);CTB oligo (C-terminal biotinylated ADDLs/globulomers); CTB mono(C-terminal biotinylated monomers); NTB mono (N-terminal biotinylatedmonomers). 6E10 (mouse mAb to Aβ amino acids 1-16; BioLegend, San Diego,CA). Oligomers Fibrils CTB oligo CTB mono NTB mono Antibody nM nM nM nMnM 3H3 40 2.1 35 3.4 240 4G1 8.8 14 11.4 16 1700 4A6 1300 11 >200016 >2000 2C10 >2000 16 >2000 >2000 >2000 6E10 69 56 50 47 158

Human mAbs were assayed by SPR for binding to aggregated islet amyloidpeptide IAPP and tau paired helical filaments (TAU PHF) isolated from ADbrain homogenates (FIG. 14). The antibody concentrations used in thisstudy were 110 nM for the IAPP and 66.7 nM for the Tau-PHF. The 3H3 and4G1 mAbs are notable for binding to Tau PHF, consistent with recognitionof a pan-amyloid epitope.

Binding of the 4G1 and 3H3 mAbs to tau40 HEK-293 and HEK-293 cell lineswas assayed (FIG. 15).

Fibrillization of Aβ42 of amyloid beta oligomers alone or in presence ofanti-amyloid mAbs, 3H3, 4G1 and 4A6 was assayed (FIG. 16).

Example 3: Use of Pan-Amyloid Antibodies as Therapeutic AntibodiesAgainst Y. pestis

A member of Enterobacteriaceae family, Yersinia pestis, is the causativeagent of the disease plague. Y. pestis infects primarily rodents, butwhich may also infect a wide array of other mammalian hosts, includinghumans. Fleas are employed by the pathogen as vectors, and the majorityof transmission events are believed to occur through flea bites. Thus,the success of the bacterium depends, in part, on its ability to adaptand quickly respond to the disparate temperature environmentsencountered in the mammalian and flea hosts. Y. pestis form biofilms inthe flea host and this trait has been associated with deadly infectionsin humans. Furthermore, biofilms of Y. pestis have also been observed inthe granulomas produced during infection. However, the nature of thesebiofilms has yet to be explored.

A number of plasmid and chromosome-encoded virulence genes have beenidentified in Y. pestis including those involved in Congo red binding(Crb+ phenotype), which forms the basis of biofilm formation. However,these studies have not yet identified an amyloid protein which serves asthe scaffold for Y. pestis biofilms.

Experiments are designed to identify and validate Yersinia amyloids thatare involved in biofilm formation during infection using a proteomicapproach and a software that has been established to identify amyloidproteins through sequence analysis. Candidate proteins are then selectedand validated. Recombinant proteins are generated and tested for theiramyloid activity using functional assays including Thioflavin T bindingassay, Congo Red Spectrometry and Electron Microscopy.

After validation of amyloids, antibodies targeting these proteins willbe generated. The mAbs generated are tested for their ability to disruptbiofilms, including biofilms of Y. pseudotuberculosis, Y. enterocoliticaand E. coli, and for their in vitro anti-biofilm activity using standardbiofilm assays. Antibodies that show a strong an anti-pan-amyloidactivity are further tested in vivo against Y. pseudotuberculosis, Y.enterocolitica and E. coli using a catheter-biofilm model. mAbs thatshow the highest activity both in vitro and in vivo activities arepotential therapeutic antibodies against Y. pestis.

Example 4: Antibody Sequences

SEQ ID NO:1: ALZ.3H3 Heavy Chain (nucleotide)SEQ ID NO:2: ALZ.3H3 Heavy Chain (amino acid)SEQ ID NO:3: ALZ.3H3 Heavy Chain variable region (nucleotide)SEQ ID NO:4: ALZ.3H3 Heavy Chain variable region (amino acid)SEQ ID NO:5: ALZ.3H3 Heavy Chain (nucleotide) with 4 nt before the Met

-   -   V-D-J-REGION: Nucleotides 62 . . . 448 of SEQ ID NO:5    -   V-REGION Nucleotides 62 . . . 357 of SEQ ID NO:5    -   FR1-IMGT (SEQ ID NO:6; SEQ ID NO:7)    -   CDR1-IMGT (SEQ ID NO:8; SEQ ID NO:9)    -   FR2-IMGT (SEQ ID NO:10; SEQ ID NO:11)    -   CDR2-IMGT (SEQ ID NO:12; SEQ ID NO:13)    -   FR3-IMGT (SEQ ID NO:14; SEQ ID NO:15)    -   CDR3-IMGT (SEQ ID NO:16; SEQ ID NO:17)    -   JUNCTION (SEQ ID NO:18; SEQ ID NO:19)    -   3′V-REGION (SEQ ID NO:20; SEQ ID NO:21)    -   N1-REGION (SEQ ID NO:22; SEQ ID NO:23)    -   D-REGION (SEQ ID NO:24; SEQ ID NO:25)    -   N2-REGION (SEQ ID NO:26; SEQ ID NO:27)    -   5′J-REGION (SEQ ID NO:28; SEQ ID NO:29)    -   J-REGION (SEQ ID NO:30; SEQ ID NO:31)    -   FR4-IMGT (SEQ ID NO:32; SEQ ID NO:33)        SEQ ID NO:34: ALZ.3H3 Light Chain (nucleotide)        SEQ ID NO:35: ALZ.3H3 Light Chain (amino acid)    -   V-J-REGION<Nucleotides 1 . . . 323 of SEQ ID NO:34    -   V-REGION<Nucleotides 1 . . . 288 of SEQ ID NO:34    -   FR1-IMGT (SEQ ID NO:36; SEQ ID NO:37)    -   CDR1-IMGT (SEQ ID NO:38; SEQ ID NO:39)    -   FR2-IMGT (SEQ ID NO:40; SEQ ID NO:41)    -   CDR2-IMGT (SEQ ID NO:42; SEQ ID NO:43)    -   FR3-IMGT (SEQ ID NO:44; SEQ ID NO:45)    -   CDR3-IMGT (SEQ ID NO:46; SEQ ID NO:47)    -   JUNCTION (SEQ ID NO:48; SEQ ID NO:49)    -   3′V-REGION (SEQ ID NO:50; SEQ ID NO:51)    -   N-REGION Nucleotides 289 . . . 290 of SEQ ID NO:34    -   5′J-REGION Nucleotides 291 . . . 295 of SEQ ID NO:34    -   J-REGION (SEQ ID NO:52; SEQ ID NO:53)    -   FR4-IMGT (SEQ ID NO:54; SEQ ID NO:55)        SEQ ID NO:56 ALZ.4A6 Heavy Chain (nucleotide)        SEQ ID NO:57 ALZ.4A6 Heavy Chain (amino acid)        SEQ ID NO:58 ALZ.4A6 Light Chain (nucleotide)        SEQ ID NO:59 ALZ.4A6 Light Chain (amino acid)        SEQ ID NO:60 ALZ.4G1 Heavy Chain (nucleotide)        SEQ ID NO:61 ALZ.4G1 Heavy Chain (amino acid)        SEQ ID NO:62 ALZ.4G1 Light Chain (nucleotide)        SEQ ID NO:63 ALZ.4G1 Light Chain (amino acid)

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A composition comprising a therapeutic antibody, wherein the antibodyis specific for binding to an epitope of curli and further wherein theepitope of curli comprises a sequence having homology to an antibodybinding site of one or more heterologous amyloid proteins.
 2. Thecomposition of claim 1, wherein the antibody inhibits fibrillization ofone or more heterologous amyloid proteins.
 3. The composition of claim1, wherein the antibody prevents biofilm formation or alters biofilmarchitecture.
 4. The composition of claim 1, wherein the antibody iseffective in reducing biofilm mass.
 5. The composition of claim 3,wherein the biofilm mass is associated with a bacteria selected from thegroup consisting of gram-positive, gram-negative, and a combinationthereof.
 6. The composition of claim 1, wherein the antibody inhibitsamyloid-β fibrillization and prevents biofilm formation or altersbiofilm architecture.
 7. The composition of claim 1, wherein theantibody is a monoclonal antibody selected from the group consisting of:a) ALZ.3H3, comprising at least one of a heavy chain amino acid sequenceas set forth in SEQ ID NO:2 and a light chain amino acid sequence as setforth in SEQ ID NO:35; b) ALZ.4G1, comprising at least one of a heavychain amino acid sequence as set forth in SEQ ID NO:61 and a light chainamino acid sequence as set forth in SEQ ID NO:63; and c) ALZ.4A6,comprising at least one of a heavy chain amino acid sequence as setforth in SEQ ID NO:57 and a light chain amino acid sequence as set forthin SEQ ID NO:59.
 8. The composition of claim 1, wherein the compositionis for application to a surface of a medical device.
 9. The compositionof claim 1, further comprising an antibiotic.
 10. The composition ofclaim 1, further comprising one or more pharmaceutically acceptablecarriers or excipients.
 11. The composition of claim 1, wherein theformulation is a topical formulation in the form of a cream, a lotion,an ointment, a hydrogel, a colloid, a gel, a foam, an oil, a milk, asuspension, a wipe, a sponge, a solution, an emulsion, a paste, a patch,a pledget, a swab, a dressing, a spray or a pad.
 12. A method ofdecolonizing a microbial organism comprising contacting the microbialorganism with a composition according to claim
 1. 13. A method ofdestroying or disrupting or inhibiting or reducing biofilm formation ofa microbial organism comprising contacting the microbial organism with acomposition according to claim
 1. 14. The method of claim 11, whereinthe microbial organism is a bacterium.
 15. A method of treating amicrobial infection in a subject comprising administering to the subjecta therapeutically effective amount of the composition of claim
 1. 16.The method of claim 15, wherein the microbial infection is a bacterialinfection.
 17. The method of claim 16, wherein the bacterial infectionis characterized by colonization of a bacterium.
 18. The method of claim17, wherein the bacterial infection is characterized by biofilmformation.
 19. The method of claim 15, wherein the microbial infectionis a topical infection.
 20. The method of claim 19, wherein the topicalinfection is selected from wound, ulcer and lesion.